Cooling loop with a supercritical fluid system using compressed refrigerant fluid flow with a positive Joule-Thomson coefficient

ABSTRACT

Provided is a chiller and system that may be utilized in a supercritical fluid chromatography method, wherein a non-polar solvent may replace a portion or all of a polar solvent for the purpose of separating or extracting desired sample molecules from a combined sample/solvent stream. The system may reduce the amount of polar solvent necessary for chromatographic separation and/or extraction of desired samples. The system may incorporate a supercritical fluid chiller, a supercritical fluid pressure-equalizing vessel and a supercritical fluid cyclonic separator. The supercritical fluid chiller allows for efficient and consistent pumping of liquid-phase gases employing off-the-shelf HPLC pumps. The pressure equalizing vessel allows the use of off-the-shelf HPLC column cartridges. The system may further incorporate the use of one or more disposable cartridges containing silica gel or other suitable medium. The system may also utilize an open loop cooling circuit using fluids with a positive Joule-Thompson coefficient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/784,131, and is a continuation of U.S. patent application Ser. No.15/504,313, filed on Feb. 15, 2017 and which is a U.S. national phaseapplication under 35 U.S.C. 371 of International Application No.PCT/US2015/044306, filed on Aug. 7, 2015, which claims the benefit under35 U.S.C. 119(e) of U.S. Provisional Application No. 62/039,066, filedon Aug. 19, 2014, U.S. Provisional Application No. 62/039,074, filed onAug. 19, 2014, and U.S. Provisional Application No. 62/039,083, filed onAug. 19, 2014; this application is also a continuation of U.S. patentapplication Ser. No. 15/397,452, filed on Jan. 3, 2017 and currentlypending, which claims the benefit of priority to U.S. ProvisionalApplication No. 62/274,659, filed on Jan. 4, 2016, U.S. ProvisionalApplication No. 62/274,667, filed on Jan. 4, 2016, U.S. ProvisionalApplication No. 62/274,672, filed on Jan. 4, 2016, U.S. ProvisionalApplication No. 62/274,748, filed on Jan. 4, 2016, U.S. ProvisionalApplication No. 62/276,102, filed on Jan. 7, 2016, and U.S. ProvisionalApplication No. 62/408,346, filed on Oct. 14, 2016; all of which areincorporated herein by reference in their entireties for all purposes.

FIELD OF THE INVENTION

Provided is a cooling loop refrigeration circuit as part of a largersystem, wherein the system requires cooling and/or thermal energytransfer.

BACKGROUND

Cooling systems utilizing the Joule-Thomson effect are well known in theart. The Joule-Thomson effect, also known as a throttling process,refers to the temperature change of a gas or liquid when allowed toexpand by passing through a valve of conduit. The temperature change isquantified by the Joule-Thomson coefficient, which may be positive(cooling) or negative (heating). Such systems are described in U.S. Pat.No. 2,991,633 to Simon, which discloses that such systems are employedwhere it is desired to obtain extremely low temperatures; Joule-Thomsoneffect cooling devices are capable of producing temperatures as low as−196° C. Simon describes the conventional device as including a thinwall tube or jacket having a closed lower end and a low pressure gasdischarge opening adjacent its other end, the jacket being formed ofsuitable material having good heat transfer properties, such asstainless steel. Simon goes on to describe, as entering the jacket, asmall elongated capillary tube extending downwardly, typically in acoiled coil configuration and terminating in a small nozzle. Simonexplains that a gas having a Joule-Thomson coefficient which is positiveat room temperature, such as nitrogen, may be supplied under highpressure to the capillary tube and will expand through the nozzle,whereby the expansion of the gas to the nozzle causes initial cooling,and the gas then flows upwardly over the convolutions of the tubing thusextracting further heat from the tubing in the nature of a heatexchanger. Eventually, the gas is exhausted to the atmosphere throughthe low pressure discharge opening of the jacket.

In application to supercritical fluid systems, however, theJoule-Thomson effect has been described in the art as an undesirablyproblem to be avoided or offset. See U.S. Pat. No. 5,653,884 to Burfordet. al. Burford explains that unwanted depressurization occurs in flowrestrictor tips in supercritical fluid systems which are designed toregulate and restrict backpressure. Specifically, Burford explains thatthe reduction in fluid density, combined with the Joule-Thomson coolingeffect, occurs at the restrictor tip which can a decrease in thesolubility of the sample/species extracted by the supercritical fluidand thus lead to unwanted precipitation and ultimately to undesirablyplugging of the restrictor. Burford explains how this undesirable effecthad been addressed to date. To avoid restrictor plugging the linearrestrictor is heated, as heating the restrictor counteracts theJoule-Thomson cooling effect at the restrictor tip and increases thesupercritical fluid solubility of the sample/species having somevolatility. Several manufacturers use a heated linear flow restrictor.

Applicants are unaware of a supercritical fluid system that utilizes thesame gas source intended for the supercritical fluidextraction/separation process as a refrigerant to be passed through acircuit with heat exchanger that does the oppose of counteract theJoule-Thompson cooling effect. Specifically, applicants are unaware of asupercritical fluid system that embraces the Joule-Thompson effect inorder to remove heat from supercritical fluid within the system.

Such a refrigeration circuit within a supercritical fluid system mayhave many applications but one such application is traditional flashchromatography.

Traditional flash chromatography is a chromatographic separationtechnique that is used to separate organic reaction mixtures to allowthe organic chemist to crudely purify the reaction products and then usethese purified products to move on to the next step of an organicsynthesis. A typical pharmaceutical synthesis has many reaction steps toget from starting materials to a final product where each of thesereaction products needs to be purified before moving on to the nextsynthetic step. The traditional flash chromatography unit employsmultiple organic solvent pumps (200 psi and 200 mls/min maximumoperation pressure and flow rate for a traditional flash chromatographyunit), a sample injection assembly where a chemist would inject thecrude reaction mix for separation, a separation column in the form of acartridge loaded with a silica or modified silica gel, a UV-VIS detector(or other form of sample detection) to detect and allow for collectionof the various fractions of the reaction mix exiting the column, and acollection tray to collect the various fractions of the reaction mixtureproducts.

Traditional flash chromatography uses large amounts of organic solvents(for example, Hexanes, Methylene Chloride, Carbon Tetra Chloride,Acetonitrile, and Chloroform) to elucidate a separation. These solventsare typically 80-90% of the flow stream through the separation column.

In the past, Applicants worked on a supercritical carbon dioxideprechiller system that included a waterless refrigeration system tosupply subcooling of liquefied carbon dioxide prior to flowing into apiston-style positive displacement pump. Since this device was created,it has undergone testing with a pump meant to supply high pressurecarbon dioxide (e.g. >100 bar) to a supercritical carbon dioxide (scCO₂)extraction system. Despite multiple attempts to improve the mechanicalbehavior of the pump, the system mass flow rates were neverproportionate to pump speed. This was indicative of cavitation effectsin the flow system comprised of duplex pump heads, each comprised of aninlet check valve, compression piston, and outlet check valve.Applicants made multiple attempts to characterize the system as afunction of inlet pressure and temperature. Despite significant effortto characterize the behavior, the pump performance was not repeatable.Moreover, all attempts at linearization via compensation failed.

In all cases, the inlet CO₂ temperature was reduced to between 2° C. and5° C. using the waterless refrigeration system. This was readily withinthe range of single stage compressor. Traditional flash chromatographyapplications have migrated to higher pressure systems to improveperformance. Higher pressure systems, however, require use of aseparation column capable of withstanding higher pressures. Traditionalflash chromatography cartridges made of plastic and intended to bedisposable are generally unusable in higher pressure (e.g., ‘mediumpressure’) flash chromatography systems.

A heretofore unsolved need exists in the industry for a system that willallow higher performance chromatography using higher solvent flow streampressures, while still allowing the use of convenient, disposableplastic cartridges prepacked for use as a chromatographic separationcolumn, e.g., pre-filled with silica gel.

SUMMARY

In one aspect, is a cooling loop refrigeration circuit as part of asupercritical fluid (or otherwise compressed fluid) system, wherein thecircuit uses compressed refrigerant fluid flow with a positiveJoule-Thompson coefficient from the refrigerant's expansion, andcommensurate temperature reduction, as it flows through an expansiondevice (e.g., capillaries, orifices and/or larger diameter channels) inthe circuit, thus allowing the refrigerant to absorb thermal energy froma source of supercritical fluid placed in proximity to the expansiondevice.

The cooling loop refrigeration circuit may be used as supercriticalfluid system designed for chemical extraction and separation processesthat use no or little solvents compared to prior art techniques such asthe system disclosed in International Application No. PCT/US2015/044306,the components of which are further detailed below.

Chiller or Pre-Chiller

In one aspect, provided is a chiller. In some embodiments, the chillercomprises: a) a first refrigerant circuit, comprising: i) a firstcompressor that pumps refrigerant through the first refrigerant circuit;ii) a first tube-in-tube heat exchanger in fluid communication with thefirst compressor, wherein the first tube-in-tube heat exchangercomprises an inner lumen and an outer lumen that surrounds the innerlumen, wherein the refrigerant flows through the outer lumen; b) acryogenic refrigerant circuit in thermodynamic communication with thefirst refrigerant circuit, the cryogenic refrigerant circuit comprising:i) a second compressor that pumps cryogenic refrigerant through the 30cryogenic refrigerant circuit; ii) the first tube-in-tube heat exchangerin fluid communication with the second compressor; wherein the cryogenicrefrigerant flows through the inner lumen; iii) a second tube-in-tubeheat exchanger in fluid communication with the first tube-in-tube heatexchanger; wherein the second tube-in-tube heat exchanger comprises aninner lumen and an outer lumen that surrounds the inner lumen, whereinthe cryogenic refrigerant flows through the outer lumen and whereinliquefied gas or supercritical gas flows through the inner lumen;wherein the chiller does not comprise an intervening medium thatmediates heat exchange between the first refrigerant circuit and thecryogenic refrigerant circuit and wherein the liquefied gas orsupercritical gas exiting the inner lumen of the second tube-in-tubeheat exchanger is chilled.

In varying embodiments, the output liquefied gas or supercritical gas ischilled at least about 35 degree C. lower than the input liquefied gasor supercritical gas. In varying embodiments, the refrigerant isselected from the group consisting of R-11, R-12, R-22, R-32, R-114,R-115, R-123, R-124, R-125, R-134A, R-142b, R-143a, R-152a, R-290,R-401A, R-401B, R-404A, R-407C, R-410A, R-409A, R-414B, R-416A, R-422B,R-422D, R-500, R-502, R-507, R-600 and mixtures thereof. In varyingembodiments, the cryogenic refrigerant is selected from the groupconsisting of R-12, R-13, R-22, R-23, R-32, R-115, R 116, R-124, R-125,R-134A, R-142b, R-143a, R-152a, R-218, R-290, R-218, R-401A, R-401B,R-402A, R-402B, R-403B, R-404A, R-408A, R-409A, R-410A, R-414B, R-416A,R-422B, R-407A, R-407C, R-408A, R-409A, R-414B, R-422A, R-422B, R-422C,R-422D, R-500, R-502, R-503, R-508B, R-507, R-508B, R-600a and mixturesthereof. In varying embodiments, the first refrigerant circuit furthercomprises in fluid communication with the first compressor and the firsttube-in-tube heat exchanger: iii) a first expansion valve; and iv) aliquid to air heat exchanger. In varying embodiments, the cryogenicrefrigerant circuit further comprises in fluid communication with thesecond compressor, the first tube-in-tube heat exchanger and the secondtube-in-tube heat exchanger: iv) a second expansion valve. In varyingembodiments, the chiller comprises a configuration as depicted in FIGS.3A-3B.

In another aspect, provided are methods of supplying a liquid-phase gasto a liquefied gas or supercritical gas extraction system. In someembodiments, the methods comprise: a) subcooling the liquid-phase gas toa temperature of −10° C. or lower; b) pumping the subcooled liquid-phasegas into a chamber configured for extraction with liquefied gas orsupercritical gas extraction, whereby the pumping mass flow rate of thesubcooled liquid phase gas is repeatable and proportionate to pumpspeed. In varying embodiments, the subcooling is performed using achiller as described above and herein. In varying embodiments, theliquefied gas or supercritical gas is selected from the group consistingof carbon dioxide, n-butane, n-propane, isobutane, dimethyl ether, andmixtures thereof. In varying embodiments, the liquefied gas orsupercritical gas is CO₂. In varying embodiments, the pumping stepemploys a positive displacement pump. In varying embodiments, thepositive displacement pump is an unmodified high performance liquidchromatography (HPLC) pump. In varying embodiments, the system furthercomprises a post-pump heater downstream of and in fluid communicationwith the pump, wherein the post-pump heater heats the liquefied gas orsupercritical gas up to an operational temperature. In varyingembodiments, the liquefied carbon dioxide is subcooled to a temperaturein the range of about −10° C. to about −40° C. In varying embodiments,the liquefied carbon dioxide is subcooled to a temperature in the rangeof about −20° C. to about −40° C. In varying embodiments, the subcoolingof the liquefied gas is performed employing a 2-stagerefrigerant-on-refrigerant chiller system. In varying embodiments, thepumping step employs a pump comprising at least one pump head and themethod does not comprise separately cooling the at least one pump head.In varying embodiments, the liquefied gas or supercritical gas ispressurized to at least about 145 psi (at least about 10 bar).

In a further aspect, provided is a system comprising a chiller asdescribed above and herein. In some embodiments, the system comprises:a) a tank comprising a gas stored at saturated conditions and a liquidwithdrawal means; b) a chiller downstream of and in fluid communicationwith the tank, wherein the chiller subcools the gas to a temperature of−10° C. or lower; c) a pump downstream of and in fluid communicationwith the tank and the chiller and a chamber configured for liquefied gasor supercritical gas extraction, wherein the pump comprises the gas at atemperature of −10° C. or lower; wherein the mass flow rate of thesubcooled liquid phase gas through the pump is repeatable andproportionate to pump speed. In some embodiments, the gas is selectedfrom the group consisting of carbon dioxide, n-butane, n-propane,isobutane, dimethyl ether, and mixtures thereof. In some embodiments,the pump is a positive displacement pump. In some embodiments, thepositive displacement pump is an unmodified high performance liquidchromatography (HPLC) pump.

In various embodiments, the system further comprises a cyclonicseparator comprising: a) a cyclone body comprising an inner surface, anouter circumference, a top outlet, a tangential inlet and a bottomoutlet, wherein the inner surface comprises a top portion, a middleportion and a bottom portion, wherein: i) the top portion of the innersurface comprises screw threads; ii) the middle portion of the innersurface is cylindrical; iii) the bottom portion of the inner surfacecomprises a funnel, wherein the funnel has an angle in the range ofabout 30 degrees to about 60 degrees; and wherein the ratio of thediameter of the outer circumference to the inner diameter of themid-height of the funnel is in the range of about 3 to about 4; and b) acap comprising a sintered filter and screw threads, wherein the screwthreads of the cap interlock with the screw threads on the inner surfaceof the top portion of the cyclone body, wherein the cyclonic separatorcan withstand pressures of at least about 1000 psi, and wherein the bodyis in fluid communication with the cap. In varying embodiments, thebottom outlet of the body of the cyclonic separator is attached to acollection container, wherein the body is in fluid communication withthe collection container. In varying embodiments, the cyclonic separatorcan withstand pressures of up to about 2000 psi. In varying embodiments,the cyclonic separator can withstand pressures of up to about 1500 psi.In varying embodiments, the thickness of the middle portion and thebottom portion of the cyclone body is at least about 0.30 inches. Invarying embodiments, the cyclone body is made of a material selectedfrom the group consisting of: stainless steel and titanium. In varyingembodiments, the stainless steel comprises an austeniticnickel-chromium-based alloy or a martensitic nickel-chromium-basedalloy. In varying embodiments, the stainless steel comprises less thanabout 0.1 wt. % carbon. In varying embodiments, the stainless steelcomprises at least a 30,000 psi yield strength. In varying embodiments,the stainless steel is selected from the group consisting of AmericanIron and Steel Institute (AISI) TYPE 304 SS, AISI TYPE 316L, INCONEL®alloy 625, INCONEL® alloy 718, AK Steel 17-4-PH®, HASTELLOY® C-22 andHASTELLOY® C 276. In varying embodiments, the stainless steel is anickel-chromium superalloy selected from the group consisting ofINCONEL® alloy 625, INCONEL® alloy 718, AK Steel 17-4-PH®, HASTELLOY®C-22 and HASTELLOY® C 276. In varying embodiments, the inner surface ofthe cyclone body is configured to induce or guide a conical cyclone offluid flowing in from the tangential inlet. In varying embodiments, theinner surface of the cyclone body does not comprise a filter or a poroussurface. In varying embodiments, the inner surface of the cyclone bodydoes not comprise one or more baffles. In varying embodiments, thecyclone body does not comprise multiple inlets. In varying embodiments,the sintered filter within the cap comprises a G-5 porosity grade (1-16microns pore size). In varying embodiments, the funnel has an angle ofabout 40 degrees; and wherein the ratio of the diameter of the outercircumference to the inner diameter of the mid-height of the funnel isabout 3.5. In varying embodiments, the bottom outlet remains open. Invarying embodiments, the cyclonic separator is as depicted in any one ofFIGS. 13 to 17. In varying embodiments, the system comprises 2 to 8cyclonic separators, e.g., 2, 3, 4, 5, 6, 7 or 8 cyclonic separators. Invarying embodiments, the interior of the cyclonic separator is in fluidconnection with atmospheric pressure.

In varying embodiments, the system further comprises a pressureequalizing vessel downstream of and in fluid communication with thechiller and the pump and upstream of and in fluid communication with thecyclonic separator, the pressure equalizing vessel comprising: i) aninner chromatography column comprising stationary phase media; and ii)an outer column that cylindrically surrounds the length of the innercolumn, wherein the interspace between the inner diameter of the outercolumn and the outer diameter of the inner chromatography columncomprises a width of at least 1 mm, wherein the outer column withstandspressures of at least about 500 psi (about 35 bar), and wherein no partof the inner column is exposed to full internal pressure withoutbalancing external equalizing pressure. In varying embodiments, theinterspace between the inner diameter of the outer column and the outerdiameter of the inner chromatography column is filled with asupercritical fluid. In varying embodiments, the inner column and theouter column can be concurrently filled with supercritical fluid under apressure in the range of about 500 psi (about 35 bar) to about 20,000psi (about 1380 bar). In varying embodiments, the inner column and theouter column can be concurrently filled with supercritical fluid under apressure in the range of at least about 5076 psi (at least about 350bar). In varying embodiments, the pressure differential across the innercolumn from top to bottom is less than the pressure rating of the innercolumn. In varying embodiments, the pressure differential between theinternal space of the inner column and the interspace is at or less thanabout 200 psi (about 14 bar). In varying embodiments, the pressurewithin the interspace is higher than the pressure within the internalspace of the inner column. In varying embodiments, the inner columncomprises an inlet end and an outlet end and the pressure at the inletend is substantially the same as the pressure at the outlet end. Invarying embodiments, the inner column is an off-the-shelf columncompatible for use in a flash chromatography system. In varyingembodiments, the inner column comprises a size in the range of fromabout 4 grams to about 350 grams stationary phase media. In varyingembodiments, the inner column comprises a diameter in the range of about0.5 inches to about 3.5 inches and a column length in the range fromabout 3.5 inches to about 11 inches. In varying embodiments, thestationary phase comprises an average particle size in the range ofabout 10 to about 100 microns. In varying embodiments, the vesselcomprises an inlet adaptor which fits to a female slip or lueR-lockconnector. In varying embodiments, the vessel comprises an outletadaptor which fits to a male slip or lueR-lock connector. In varyingembodiments, the outlet adaptor comprises an O-ring that seals aroundthe male slip or lueR-lock connector. In varying embodiments, the innercolumn comprises an inlet end and an outlet end, wherein neither theinlet end nor the outlet end of the inner column comprises a perforatedstopper. In varying embodiments, the interspace comprises a single inletand no outlet or vent. In varying embodiments, the pressure equalizingcolumn is as depicted in FIGS. 11 and 12.

In varying embodiments, the liquefied gas or supercritical gas is CO₂.In varying embodiments, the liquefied gas or supercritical gas ispressurized to at least about 145 psi (10 bar). In varying embodiments,the flow of the supercritical solvent through the system is in the rangeof about 10 ml/min, e.g., at least about 15 ml/min, 20 ml/min, 25ml/min, 30 ml/min, 35 ml/min, 40 ml/min, 45 ml/min, or 50 ml/min, toabout 300 ml/min. In varying embodiments, the system further pumps aco-solvent. In some embodiments, the co-solvent comprises an alcohol ofthree or fewer carbon atoms (e.g., methanol, ethanol, propanol,isopropanol) or an acetate of three or fewer carbon atoms (e.g., methylacetate, ethyl acetate, propyl acetate), or mixtures thereof. In varyingembodiments, the system is as depicted in FIG. 1.

In another aspect, provided are methods of performing high pressureseparation and/or extraction procedures using a flash chromatographysystem. In some embodiments, the methods comprise inputting a stream ofgas phase supercritical fluid comprising molecules into a chiller asdescribed above and herein.

Pressure Equalizing Vessel

In one aspect, provided is a pressure equalizing chromatography vesselcomprising:

i) an inner chromatography column comprising stationary phase media; and

ii) an outer column that cylindrically surrounds the length of the innercolumn, wherein the interspace between the inner diameter of the outercolumn and the outer diameter of the inner chromatography columncomprises a width of at least 1 mm, wherein the outer column withstandspressures of at least about 500 psi (about 35 bar), and wherein no partof the inner column is exposed to full internal pressure withoutbalancing external equalizing pressure. In varying embodiments, theinterspace between the inner diameter of the outer column and the outerdiameter of the inner chromatography column is filled with asupercritical fluid. In varying embodiments, the inner column and theouter column can be concurrently filled with supercritical fluid under apressure in the range of about 500 psi (about 35 bar) to about 20,000psi (about 1380 bar). In varying embodiments, the inner column and theouter column can be concurrently filled with supercritical fluid under apressure of at least about 5076 psi (at least about 350 bar). In varyingembodiments, the pressure differential across the inner column from topto bottom is less than the pressure rating for the inner column. Invarying embodiments, the pressure differential between the internalspace of the inner column and the interspace is at or less than about200 psi (about 14 bar). In varying embodiments, the pressure within theinterspace is higher than the pressure within the internal space of theinner column. In varying embodiments, the inner column comprises aninlet end and an outlet end and the pressure at the inlet end issubstantially the same as the pressure at the outlet end. In varyingembodiments, the inner column is an off-the-shelf column compatible foruse in a flash chromatography system. In varying embodiments, the innercolumn comprises a size in the range of from about 4 grams to about 350grams stationary phase media. In varying embodiments, the inner columncomprises a diameter in the range of about 0.5 inches to about 3.5inches and a column length in the range from about 3.5 inches to about11 inches. In varying embodiments, the stationary phase comprises anaverage particle size in the range of about 10 to about 100 microns. Invarying embodiments, the vessel comprises an inlet adaptor which fits toa female slip or lueR-lock connector. In varying embodiments, the vesselcomprises an outlet adaptor which fits to a male slip or lueR-lockconnector. In varying embodiments, the outlet adaptor comprises anO-ring that seals around the male slip or lueR-lock connector. Invarying embodiments, the inner column comprises an inlet end and anoutlet end, wherein neither the inlet end nor the outlet end of theinner column comprises a perforated stopper. In varying embodiments, theinterspace comprises a single inlet and no outlet or vent. In varyingembodiments, the pressure equalizing column is as depicted in FIGS. 11and 12.

In another aspect, provided is a chromatography system comprising thepressure equalizing vessel as described above and herein, wherein thesystem is pressurized and pumps a supercritical solvent. In someembodiments, the system further comprises a supercritical solvent pumpupstream of and in fluid communication with the pressure equalizingvessel and a chiller upstream of and in fluid communication with thepump, wherein the chiller reduces the temperature of the supercriticalsolvent to about −5° C. or lower, e.g., about −10° C., −15° C., −20° C.,−25° C., or lower. In some embodiments, the chiller comprises: a) afirst refrigerant circuit, comprising: i) a first compressor that pumpsrefrigerant through the first refrigerant circuit; ii) a firsttube-in-tube heat exchanger in fluid communication with the firstcompressor, wherein the first tube-in-tube heat exchanger comprises aninner lumen and an outer lumen that surrounds the inner lumen, whereinthe refrigerant flows through the outer lumen; b) a cryogenicrefrigerant circuit in thermodynamic communication with the firstrefrigerant circuit, the cryogenic refrigerant circuit comprising: i) asecond compressor that pumps cryogenic refrigerant through the cryogenicrefrigerant circuit; ii) the first tube-in-tube heat exchanger in fluidcommunication with the second compressor; wherein the cryogenicrefrigerant flows through the inner lumen; iii) a second tube-in-tubeheat exchanger in fluid communication with the first tube-in-tube heatexchanger; wherein the second tube-in-tube heat exchanger comprises aninner lumen and an outer lumen that surrounds the inner lumen, whereinthe cryogenic refrigerant flows through the outer lumen and whereinliquefied gas or supercritical gas flows through the inner lumen;wherein the chiller does not comprise an intervening medium thatmediates heat exchange between the first refrigerant circuit and thecryogenic refrigerant circuit and wherein the liquefied gas orsupercritical gas exiting the inner lumen of the second tube-in-tubeheat exchanger is chilled. In varying embodiments, the output liquefiedgas or supercritical gas is chilled at least about 35° C. lower than theinput liquefied gas or supercritical gas. In varying embodiments, therefrigerant is selected from the group consisting of R-11, R-12, R-22,R-32, R-114, R-115, R-123, R-124, R-125, R-134A, R-142b, R-143a, R-152a,R-290, R-401A, R-401B, R-404A, R-407C, R-410A, R-409A, R-414B, R-416A,R-422B, R-422D, R-500, R-502, R-507, R-600 and mixtures thereof. Invarying embodiments, the cryogenic refrigerant is selected from thegroup consisting of R-12, R-13, R-22, R-23, R-32, R-115, R-116, R-124,R-125, R-134A, R-142b, R-143a, R-152a, R-218, R-290, R-218, R-401A,R-401B, R-402A, R-402B, R-403B, R-404A, R-408A, R-409A, R-410A, R-414B,R-416A, R-422B, R-407A, R-407C, R-408A, R-409A, R-414B, R-422A, R-422B,R-422C, R-422D, R-500, R-502, R-503, R-508B, R-507, R-508B, R-600a andmixtures thereof. In varying embodiments, the first refrigerant circuitfurther comprises in fluid communication with the first compressor andthe first tube-in-tube heat exchanger: iii) a first expansion valve; andiv) a liquid to air heat exchanger. In varying embodiments, thecryogenic refrigerant circuit further comprises in fluid communicationwith the second compressor, the first tube-in-tube heat exchanger andthe second tube-in-tube heat exchanger: iv) a second expansion valve. Invarying embodiments, the chiller comprises a configuration as depictedin FIGS. 3A-3B. In some embodiments, the system comprises: a) a tankcomprising a gas stored at saturated conditions and a liquid withdrawalmeans; b) a chiller in fluid communication with the tank, wherein thechiller subcools the gas to a temperature of −10° C. or lower; c) a pumpin fluid communication with the tank and a chamber configured forliquefied gas or supercritical gas extraction, wherein the pumpcomprises the gas at a temperature of −10° C. or lower; wherein the massflow rate of the subcooled liquid phase gas through the pump isrepeatable and proportionate to pump speed. In some embodiments, the gasis selected from the group consisting of carbon dioxide, n-butane,n-propane, isobutane, dimethyl ether, and mixtures thereof. In someembodiments, the pump is a positive displacement pump. In someembodiments, the positive displacement pump is an unmodified highperformance liquid chromatography (HPLC) pump. In varying embodiments,the system further comprises a post-pump heater downstream of and influid communication with the pump, wherein the post-pump heater heatsthe liquefied gas or supercritical gas up to an operational temperature.

In varying embodiments, the supercritical fluid is CO₂. In varyingembodiments, the flow of the supercritical solvent through the system isin the range of about 10 ml/min, e.g., at least about 15 ml/min, 20ml/min, 25 ml/min, 30 ml/min, 35 ml/min, 40 ml/min, 45 ml/min, or 50ml/min, to about 300 ml/min. In varying embodiments, the system furtherpumps a co-solvent. In some embodiments, the co-solvent comprises analcohol of 3 or fewer carbon atoms (e.g., methanol, ethanol, propanol,isopropanol) or an acetate of 3 or fewer carbon atoms (e.g., methylacetate, ethyl acetate, propyl acetate), or mixtures thereof. In varyingembodiments, the system is as depicted in FIG. 1.

In varying embodiments, the system further comprises a cyclonicseparator downstream of and in fluid communication with the pressureequalizing vessel, the cyclonic separator comprising:

a) a cyclone body comprising an inner surface, an outer circumference, atop outlet, a tangential inlet and a bottom outlet, wherein the innersurface comprises a top portion, a middle portion and a bottom portion,wherein: i) the top portion of the inner surface comprises screwthreads; ii) the middle portion of the inner surface is cylindrical;iii) the bottom portion of the inner surface comprises a funnel, whereinthe funnel has an angle in the range of about 30 degrees to about 60degrees; and wherein the ratio of the diameter of the outercircumference to the inner diameter of the mid-height of the funnel isin the range of about 3 to about 4; and b) a cap comprising a sinteredfilter and screw threads, wherein the screw threads of the cap interlockwith the screw threads on the inner surface of the top portion of thecyclone body, wherein the cyclonic separator can withstand pressures ofat least about 1000 psi, and wherein the body is in fluid communicationwith the cap. In varying embodiments, the bottom outlet of the body isattached to a collection container, wherein the body is in fluidcommunication with the collection container. In some embodiments, thecyclonic separator can withstand pressures of up to about 10,000 psi,e.g., up to about 5000 psi, e.g., up to about 2000 psi, e.g., up toabout 1900 psi, 1800 psi, 1700 psi, 1600 psi, or 1500 psi. In varyingembodiments, the thickness of the middle portion and the bottom portionof the cyclone body is at least about 0.30 inches, e.g., at least about0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.375, 0.38, 0.39, 0.40inches. In varying embodiments, the cyclone body is made of a materialselected from the group consisting of: stainless steel and titanium. Invarying embodiments, the stainless steel comprises an austeniticnickel-chromium-based alloy or a martensitic nickel-chromium-basedalloy. In varying embodiments, the stainless steel comprises less thanabout 0.1 wt. % carbon. In varying embodiments, the stainless steelcomprises at least a 30,000 psi yield strength. In varying embodiments,the stainless steel is selected from the group consisting of AmericanIron and Steel Institute (AISI) TYPE 304 SS, AISI TYPE 316L, INCONEL®.alloy 625, INCONEL® alloy 718, AK Steel 17-4-PH®, HASTELLOY® C-22 andHASTELLOY® C-276. In varying embodiments, the stainless steel is anickel-chromium superalloy selected from the group consisting ofINCONEL® alloy 625, INCONEL® alloy 718, HASTELLOY® C-22 and HASTELLOY®C-276. In some embodiments, the inner surface of the cyclone body isconfigured to induce or guide a conical cyclone of fluid flowing in fromthe tangential inlet. In varying embodiments, the inner surface of thecyclone body does not comprise a filter or a porous surface. In varyingembodiments, the inner surface of the cyclone body does not comprise oneor more baffles. In some embodiments, the cyclone body does not comprisemultiple inlets. In some embodiments, the sintered filter within the capcomprises a G-5 porosity grade (1-16 microns pore size). In someembodiments, the funnel has an angle of about 40 degrees; and the ratioof the diameter of the outer circumference to the inner diameter of themid-height of the funnel is about 3.5. In varying embodiments, thebottom outlet remains open. In some embodiments, the cyclonic separatoris as depicted in any one of FIGS. 13 to 17.

In a further aspect, provided is a chromatography system comprising oneor more cyclonic separators as described above and herein, wherein thechromatography system is pressurized and pumps a supercritical solvent.In varying embodiments, the system comprises 2 to 8 cyclonic separators,e.g., 2, 3, 4, 5, 6, 7 or 8 cyclonic separators. In varying embodiments,the interior of the cyclonic separator is in fluid connection withatmospheric pressure.

In another aspect, provided are methods of performing high pressureseparation and/or extraction procedures using a flash chromatographysystem, comprising separating sample in a supercritical fluid mobilephase in the inner chromatography column of the pressure equalizingvessel as described above and herein.

Cyclonic Separator

In one aspect, provided is a cyclonic separator comprising: a) a cyclonebody comprising an inner surface, an outer circumference, a top outlet,a tangential inlet and a bottom outlet, wherein the inner surfacecomprises a top portion, a middle portion and a bottom portion, wherein:i) the top portion of the inner surface comprises screw threads; ii) themiddle portion of the inner surface is cylindrical; iii) the bottomportion of the inner surface comprises a funnel, wherein the funnel hasan angle in the range of about 30 degrees to about 60 degrees; andwherein the ratio of the diameter of the outer circumference to theinner diameter of the mid-height of the funnel is in the range of about3 to about 4; and b) a cap comprising a sintered filter and screwthreads, wherein the screw threads of the cap interlock with the screwthreads on the inner surface of the top portion of the cyclone body,wherein the cyclonic separator can withstand pressures of at least about1000 psi, and wherein the body is in fluid communication with the cap.In varying embodiments, the bottom outlet of the body is attached to acollection container, wherein the body is in fluid communication withthe collection container. In some embodiments, the cyclonic separatorcan withstand pressures of up to about 10,000 psi, e.g., up to about5000 psi, e.g., up to about 2000 psi, e.g., up to about 1900 psi, 1800psi, 1700 psi, 1600 psi, or 1500 psi. In varying embodiments, thethickness of the middle portion and the bottom portion of the cyclonebody is at least about 0.30 inches, e.g., at least about 0.31, 0.32,0.33, 0.34, 0.35, 0.36, 0.37, 0.375, 0.38, 0.39, 0.40 inches. In varyingembodiments, the cyclone body is made of a material selected from thegroup consisting of: stainless steel and titanium. In varyingembodiments, the stainless steel comprises an austeniticnickel-chromium-based alloy or a martensitic nickel-chromium-basedalloy. In varying embodiments, the stainless steel comprises less thanabout 0.1 wt. % carbon. In varying embodiments, the stainless steelcomprises at least a 30,000 psi yield strength. In varying embodiments,the stainless steel is selected from the group consisting of AmericanIron and Steel Institute (AISI) TYPE 304 SS, AISI TYPE 316L, INCONEL®alloy 625, INCONEL® alloy 718, AK Steel 17-4-PH®, HASTELLOY® C-22 andHASTELLOY® C-276. In varying embodiments, the stainless steel is anickel-chromium superalloy selected from the group consisting ofINCONEL® alloy 625, INCONEL® alloy 718, HASTELLOY® C-22 and HASTELLOY®C-276. In some embodiments, the inner surface of the cyclone body isconfigured to induce or guide a conical cyclone of fluid flowing in fromthe tangential inlet. In varying embodiments, the inner surface of thecyclone body does not comprise a filter or a porous surface. In varyingembodiments, the inner surface of the cyclone body does not comprise oneor more baffles. In some embodiments, the cyclone body does not comprisemultiple inlets. In some embodiments, the sintered filter within the capcomprises a G-5 porosity grade (1-16 microns pore size). In someembodiments, the funnel has an angle of about 40 degrees; and the ratioof the diameter of the outer circumference to the inner diameter of themid-height of the funnel is about 3.5. In varying embodiments, thebottom outlet remains open. In some embodiments, the cyclonic separatoris as depicted in any one of FIGS. 13 to 17.

In a further aspect, provided is a chromatography system comprising oneor more cyclonic separators as described above and herein, wherein thechromatography system is pressurized and pumps a supercritical solvent.In varying embodiments, the system comprises 2 to 8 cyclonic separators,e.g., 2, 3, 4, 5, 6, 7 or 8 cyclonic separators. In varying embodiments,the interior of the cyclonic separator is in fluid connection withatmospheric pressure.

In varying embodiments, the chromatography system further comprises apressure equalizing vessel upstream of and in fluid communication withthe cyclonic separator, the pressure equalizing vessel comprising: i) aninner chromatography column comprising stationary phase media; and ii)an outer column that cylindrically surrounds the length of the innercolumn, wherein the interspace between the inner diameter of the outercolumn and the outer diameter of the inner chromatography columncomprises a width of at least 1 mm, wherein the outer column withstandspressures of at least about 500 psi (about 35 bar), and wherein no partof the inner column is exposed to full internal pressure withoutbalancing external equalizing pressure. In varying embodiments, theinterspace has a width of up to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 mm. In varying embodiments of thepressure equalizing vessel, the interspace between the inner diameter ofthe outer column and the outer diameter of the inner chromatographycolumn is filled with a supercritical fluid. In some embodiments, theinner column and the outer column can be concurrently filled withsupercritical fluid under a pressure in the range of about 500 psi(about 35 bar) to about 20,000 psi (about 1380 bar). In someembodiments, the inner column and the outer column can be concurrentlyfilled with supercritical fluid under a pressure in the range of atleast about 5076 psi (at least about 350 bar). In some embodiments, thepressure differential across the inner column from top to bottom is lessthan the pressure rating of the inner column. Generally, the pressuredifferential between the internal space of the inner column and theinterspace is less than the pressure rating of the inner column. In someembodiments, the pressure differential between the internal space of theinner column and the interspace is at or less than about 200 psi (about14 bar). In some embodiments, the pressure within the interspace ishigher than the pressure within the internal space of the inner column.In some embodiments, the inner column comprises an inlet end and anoutlet end and the pressure at the inlet end is substantially the sameas the pressure at the outlet end. In some embodiments, the inner columnis an off-the-shelf column compatible for use in a flash chromatographysystem. In some embodiments, the inner column comprises a size in therange of from about 4 grams to about 350 grams stationary phase media.In some embodiments, the inner column comprises a diameter in the rangeof about 0.5 inches to about 3.5 inches and a column length in the rangefrom about 3.5 inches to about 11 inches. In some embodiments, thestationary phase comprises an average particle size in the range ofabout 10 to about 100 microns, e.g., in the range of about 20 to about80 microns. In some embodiments, the pressure equalizing vesselcomprises an inlet adaptor which fits to a female slip or lueR-lockconnector. In some embodiments, the pressure equalizing vessel comprisesan outlet adaptor which fits to a male slip or lueR-lock connector. Insome embodiments, the outlet adaptor comprises an O-ring that sealsaround the male slip or lueR-lock connector. In some embodiments, theinner column comprises an inlet end and an outlet end, wherein neitherthe inlet end nor the outlet end of the inner column comprises aperforated stopper. In some embodiments, the interspace comprises asingle inlet and no outlet or vent. In some embodiments, the pressureequalizing column is as depicted in FIGS. 11 and 12.

In varying embodiments, the chromatography system further comprises asupercritical solvent pump and a chiller in fluid communication with andupstream of the pump and the cyclonic separator, wherein the chillerreduces the temperature of the supercritical solvent to about −5° C. orlower, e.g., about −10° C., −15° C., −20° C., −25° C. or lower.

In some embodiments, the chiller comprises: a) a first refrigerantcircuit, comprising: i) a first compressor that pumps refrigerantthrough the first refrigerant circuit; ii) a first tube-in-tube heatexchanger in fluid communication with the first compressor, wherein thefirst tube-in-tube heat exchanger comprises an inner lumen and an outerlumen that surrounds the inner lumen, wherein the refrigerant flowsthrough the outer lumen; b) a cryogenic refrigerant circuit inthermodynamic communication with the first refrigerant circuit, thecryogenic refrigerant circuit comprising: i) a second compressor thatpumps cryogenic refrigerant through the cryogenic refrigerant circuit;ii) the first tube-in-tube heat exchanger in fluid communication withthe second compressor; wherein the cryogenic refrigerant flows throughthe inner lumen; iii) a second tube-in-tube heat exchanger in fluidcommunication with the first tube-in-tube heat exchanger; wherein thesecond tube-in-tube heat exchanger comprises an inner lumen and an outerlumen that surrounds the inner lumen, wherein the cryogenic refrigerantflows through the outer lumen and wherein liquefied gas or supercriticalgas flows through the inner lumen; wherein the chiller does not comprisean intervening medium that mediates heat exchange between the firstrefrigerant circuit and the cryogenic refrigerant circuit and whereinthe liquefied gas or supercritical gas exiting the inner lumen of thesecond tube-in-tube heat exchanger is chilled. In varying embodiments,the output liquefied gas or supercritical gas is chilled at least about35° C. lower than the input liquefied gas or supercritical gas. Invarying embodiments, the refrigerant is selected from the groupconsisting of R-11, R-12, R-22, R-32, R-114, R-115, R-123, R-124, R-125,R-134A, R-142b, R-143a, R-152a, R-290, R-401A, R-401B, R-404A, R-407C,R-410A, R-409A, R-414B, R-416A, R-422B, R-422D, R-500, R-502, R-507,R-600 and mixtures thereof. In varying embodiments, the cryogenicrefrigerant is selected from the group consisting of R-12, R-13, R-22,R-23, R-32, R-115, R-116, R-124, R-125, R-134A, R-142b, R-143a, R-152a,R-218, R-290, R-218, R-401A, R-401B, R-402A, R-402B, R-403B, R-404A,R-408A, R-409A, R-410A, R-414B, R-416A, R-4228, R-407A, R-407C, R-408A,R-409A, R-414B, R-422A, R-422B, R-422C, R-422D, R-500, R-502, R-503,R-5088, R-507, R-5088, R-600a and mixtures thereof. In varyingembodiments, the first refrigerant circuit further comprises in fluidcommunication with the first compressor and the first tube-in-tube heatexchanger: iii) a first expansion valve; and iv) a liquid to air heatexchanger. In varying embodiments, the cryogenic refrigerant circuitfurther comprises in fluid communication with the second compressor, thefirst tube-in-tube heat exchanger and the second tube-in-tube heatexchanger: iv) a second expansion valve. In varying embodiments, thechiller comprises a configuration as depicted in FIGS. 3A-3B. In someembodiments, the system comprises: a) a tank comprising a gas stored atsaturated conditions and a liquid withdrawal means; b) a chiller influid communication with the tank, wherein the chiller subcools the gasto a temperature of −10° C. or lower; c) a pump downstream of and influid communication with the tank and the chiller, and a chamberconfigured for liquefied gas or supercritical gas extraction, whereinthe pump comprises the gas at a temperature of −10° C. or lower; whereinthe mass flow rate of the subcooled liquid phase gas through the pump isrepeatable and proportionate to pump speed. In some embodiments, the gasis selected from the group consisting of carbon dioxide, n-butane,n-propane, isobutane, dimethyl ether, and mixtures thereof. In someembodiments, the pump is a positive displacement pump. In someembodiments, the positive displacement pump is an unmodified highperformance liquid chromatography (HPLC) pump. In varying embodiments,the system further comprises a post-pump heater downstream of and influid communication with the pump, wherein the post-pump heater heatsthe liquefied gas or supercritical gas up to an operational temperature.

In varying embodiments, the supercritical fluid is CO₂. In varyingembodiments, the flow of the supercritical solvent through the system isin the range of about 10 ml/min, e.g., at least about 15 ml/min, 20ml/min, 25 ml/min, 30 ml/min, 35 ml/min, 40 ml/min, 45 ml/min, or 50ml/min, to about 300 ml/min. In varying embodiments, the system furtherpumps a co-solvent. In some embodiments, the co-solvent comprises analcohol of three or fewer carbon atoms (e.g., methanol, ethanol,propanol, isopropanol) or an acetate of three or fewer carbon atoms(e.g., methyl acetate, ethyl acetate, propyl acetate), or mixturesthereof. In varying embodiments, the system is as depicted in FIG. 1.

In a related aspect, provided are methods of separating molecules from asupercritical fluid. In varying embodiments, the methods compriseinputting a stream of gas phase supercritical fluid comprising moleculesinto the tangential inlet of a cyclonic separator as described above andherein, wherein the stream of supercritical fluid rotates around theinner surface of the cyclone body, wherein the molecules separate fromthe stream, slide down the inner surface and exit the cyclone body intothe collection container; and wherein the gas phase supercritical fluidexits through the cap, and wherein any molecules still in the fluidstream do not escape through the sintered filter of the cap. In varyingembodiments, the interior of the cyclonic separator is in fluidconnection with atmospheric pressure.

Definitions

The phrase “conical cyclone of fluid” refers to a downward spiral pathwhich substantially does not cross itself.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 illustrates a flow schematic of a chromatography system describedherein.

FIG. 2 illustrates an apparatus schematic of a chromatography systemdescribed herein.

FIGS. 3A-B. FIG. 3A illustrates a block diagram of the chiller's twocircuit cascade refrigeration system. FIG. 3B illustrates the chillerand HPLC pumps package. The post pump heater is used to bring processfluids up to operational temperatures.

FIG. 4 illustrates a plot of mass flow rate vs. temperature at a set RPMof 250.

FIG. 5 illustrates a plot of mass flow rate vs. temperature at a set RPMof 500.

FIG. 6 illustrates a plot of mass flow rate vs. temperature at a set RPMof 750.

FIG. 7 illustrates trend lines for mass flow vs. temperature at the setRPMs of 250, 500 and 750.

FIG. 8 illustrates an assembly drawing of external views of a productionprototype of the chiller.

FIG. 9 illustrates an assembly drawing of internal views of a productionprototype of the chiller.

FIG. 10 illustrates an assembly drawing of internal views of aproduction prototype of the chiller.

FIG. 11 illustrates a cross sectional view of the pressure equalizingvessel showing the outer pressure containment vessel and the innerchromatography cartridge or column. Insets depict the input and outputattachments of the inner chromatography cartridge or column to the outerpressure containment vessel.

FIG. 12 illustrates a cross sectional view of the pressure containmentvessel in the context of its fluid connections with an exemplarychromatography system.

FIG. 13 illustrates an assembly drawing of the system, including abreakdown of the parts and quantities required.

FIG. 14 illustrates a CFD visualization of the streamlines of the gasflow.

FIG. 15 illustrates a manufacturing print of the Collection CycloneBody.

FIG. 16 illustrates a manufacturing print for the Collection CycloneCap.

FIG. 17 illustrates a detailed internal geometry of the CollectionCyclone Assembly.

FIG. 18 illustrates a separation of aceptophenone and methyl parabenusing the chromatography system described herein.

FIG. 19 illustrates a separation of aceptophenone and methyl parabenusing the chromatography system described herein.

FIG. 20 illustrates a separation of benzoic acid and 4-acetamidophenolusing the chromatography system described herein.

FIG. 21 illustrates a separation of ketoprofen and 4-acetamidophenolusing the chromatography system described herein.

FIG. 22 illustrates a schematic layout of a medium pressure flashchromatography system according to a preferred embodiment of theinvention.

FIG. 23 illustrates a cross sectional view of a pressure equalizationsystem, i.e., pressure containment assembly according to a preferredembodiment of the invention.

FIG. 24 illustrates a cooling system with an expansion device designedto impart fluid expansion to effectuate heat transfer where needed inthe system.

FIG. 25 illustrates an open loop cooling circuit for the refrigerant,wherein the refrigerant flows from a compressor through an inlet heatexchanger before passing to the first tube-in-tube heat exchanger.

FIG. 26 shows a graph of exemplary refrigerants having a positive JouleThomson coefficients.

FIG. 27 shows a cross-sectional view of an exemplary primary heatexchanger incorporated into a pump.

DETAILED DESCRIPTION

1. Introduction

Provided herein is gas/fluid low refrigerant circuit that may be used asa cooling loop for chilling a supercritical fluid within a supercriticalfluid system is used for extracting and separating components of achemical mixture. As described in more detail below, the cooling systemmay be comprised of an expansion device to expand a refrigerant fluidwith a positive Joule-Thomson coefficient from an area of high pressureto one of lower pressure without capturing or recompressing therefrigerant fluid. Such expansion may be accomplished by passing therefrigerant fluid through an expansion device with one or more orificesor capillaries. The expansion device may be placed in close proximity towhere the cooling effect is needed, for example, adjacent heat exchangerregion of the supercritical fluid system in order to remove thermalenergy from the supercritical fluid. The expanded refrigerant fluid maybe passed through larger diameter channels to increase the residencetime and surface area and allow the cold low pressure refrigerant fluidto absorb energy from the object being cooled, e.g., proximalsupercritical fluid. The larger diameter channels can be machineddirectly into the adjacent system to be cooled for superior heattransfer. Further efficiencies can be obtained by using the low pressurerefrigerant fluid as it is exhausted and has approached temperatureequilibrium with the object being cooled (e.g., supercritical fluid) tocool the incoming high pressure refrigerant fluid before expansion(e.g., heat exchanger) to reduce its enthalpy. This can be accomplishedwith a counter flow or other heat exchanger. The amount of coolingand/or temperature of the device being cooled can be controlled usingfeedback from a temperature sensor placed on or adjacent to the deviceor system being cooled. A control valve connected to the sensor may beused to control the flow into the pressure reduction device. Thiscooling method and systems utilizing this method will have numerousadvantages such as very compact, low noise, low equipment cost, lowoperating cost, and the ability to reach lower temperatures that manyconventional refrigeration systems.

One such supercritical fluid system that may benefit significantly fromthis improved cooling method is a supercritical fluid chromatographysystem. Specifically, the system disclosed herein is one that enable theseparation cartridges employed in traditional flash chromatographyapplications to be used in conjunction with a liquefied gas orsupercritical fluid dominated solvent system. This facilitates or allowsfor a substantial reduction on the order 80-90% of the organic solventsor a complete elimination of organic solvents in the separation process.Achieving this goal involves implementation of one or more features toenable operating conditions in the range of pressures associated withsubcritical fluids or subcritical fluids, e.g., in a pressure range ofabout 35 bar or higher pressure. These features include a prechillersystem, a pressure equalizing vessel, and a pressurized cyclonicseparator. When used in coordination, these improvements allow forliquefied gas and supercritical fluids to be utilized where only lowpressure liquid solvents could previously been employed. The prechillersystem enables a standard HPLC pump, nominally optimized for operationwith incompressible fluids, to be employed with a liquefied gas orsupercritical fluid. The pressure equalizing vessel enables anoff-the-shelf chromatography cartridge, nominally intended for use withlow pressure liquid solvents, to be used without further alteration in aliquefied gas or supercritical fluid system. The high pressure cyclonicseparator enables product recovery from a high pressure system andserves the purpose of a collection flask in a high pressure system.

2. Supercritical Fluid Chromatography Systems

The chromatography systems described herein are based, in part, on thediscovery and advanced design of traditional flash chromatographytechnology that employs supercritical fluid (e.g., liquid phase CO₂) asthe main non-polar solvent in flash chromatography. FIGS. 1 and 2illustrate a supercritical fluid CO₂ flash chromatography apparatus. Invarying embodiments, the apparatus has one or more ofprechiller/supercritical fluid (e.g., CO₂) pump that allows for theefficient and accurate delivery of liquid-phase supercritical fluid(e.g., CO₂) in a supercritical fluid state to the apparatus up to 10,000psi, e.g., up to 5000 psi or 2500 psi and at a flow rate of at leastabout 10 mls/min and up to about 250 mls/min or 300 mls/min, a secondaryco-solvent pumping package that allows for polar modifier solvents(e.g., co-solvents) to be added to the flow stream in an isocratic orgradient mode (2500 psi and 100 mls/min), an injection manifold that cantake the form of an injection loop or a secondary injection column forlarger sample injection, a pressure equalizing vessel assembly thatallows traditional flash column cartridges to be used in the apparatusup to the pressures of operation, a UV-VIS detector (other detectorsoptional), and a back pressure regulator (BPR) upstream of and in fluidcommunication with a stream selector, which is in fluid communicationwith the one or more cyclonic separators. The BPR brings the pressure ofthe flow stream from operation pressures down to ambient pressures forfraction collection in the cyclonic separators.

As shown in FIG. 1 a chromatography system 100 is pressurized to pumpsupercritical fluid 120 (e.g., CO₂), from a source 122 with or withoutco-solvent. In varying embodiments, the system further pumps aco-solvent 130 from a source. When pumping a supercritical fluid mixedwith a co-solvent, the co-solvent may comprise up to about 20% v/v ofthe fluid being pumped through the system. As shown in FIG. 1, theco-solvent is delivered through an input pump 136 separate from thesupercritical fluid input pump 126, and mixed with the supercriticalfluid in a mixer 140 prior to delivery to the inner column of thepressure equalizing vessel. In some embodiments, the co-solventcomprises an alcohol of three or fewer carbon atoms (e.g., methanol,ethanol, propanol, isopropanol) or an acetate of three or fewer carbonatoms (e.g., methyl acetate, ethyl acetate, propyl acetate), or mixturesthereof. The chromatography system shown in FIG. 1 also comprises achiller 124 and a pre-heater 128 for the supercritical fluid 120. A postheater 160, a cyclone collector 170 and a stream selector that iscapable of selecting 1 or 8 cyclone collectors for the sample.

3. Chiller for Pumping Supercritical and Liquefied Gases

A system for super chilling liquid gases (e.g., including carbondioxide, methane, ethane, propane, butane, ethylene, propylene, andethers) and to increase pumping efficiency and consistency is provided.The chiller 124 cools liquid gases (e.g., including carbon dioxide,methane, ethane, propane, butane, ethylene, propylene, and ethers) tobetween −10° C. and −40° C. and has been shown to enable the use of astandard HPLC pump with increased mass flow rates at a constant setpoint as the temperature is reduced. The herein described system reducesthe cost of pumping CO₂ by allowing the use of traditional HPLC pumps,rather than highly specialized CO₂ pumps.

The present systems and methods use a cascade chiller to cool the liquidCO₂ to less than −10° C. e.g., in varying embodiments, less than −20°C., to minimize the variance in pump performance. The lower temperaturesenable greater tolerance for the flow path in the high pressure CO₂ pumphead and facilitate the use of an unmodified HPLC (High Pressure LiquidChromatography Pump). Traditional HPLC pumps are normally intended forpumping liquids; not liquid gases. There is a wide scatter in flowperformance that results when the liquid is chilled to only 0° C. Atroom temperature conditions the variance would be greater than 30% andwould render the unmodified HPLC pump completely ineffective insupercritical chromatography applications. The herein described chillerand methods allows the use of a traditional HPLC for precise meteredpumping of liquid-gases, e.g., for delivery to extractors, reactors andchromatography equipment.

We have determined that extreme subcooling much improved pumpingperformance. The pumping mass flow rate was linearly related to speedand repeatability. In this case, the supercritical CO₂ was subcooledfrom an ambient condition of nearly 25° C. and 52 bar to approximately−25° C. and 52 bar. This 55° C. temperature reduction resulted in theliquid CO₂ conditions more closely resembling an incompressible fluid,such as water. A completely unmodified and standard HPLC pump can thenbe used to pump the scCO₂ under very linear conditions. Such behavior ishighly desirable for applications including supercritical fluidextraction, supercritical fluid solid phase extraction, supercriticalfluid flash chromatography, and supercritical fluid chromatography.

The use of this hook of physics is advantageous in these applications,as it enables standard and cost-effective HPLC pumps to be used insupercritical fluid applications with highly linearly mass flow rateswithout the need for either elaborate compensation algorithms, sensorfeedback systems involving compensation via a loss in weight measurementof the supply cylinder, direct compensation via a mass flow measure(e.g. coriolis mass meter), or the need for a booster pump to stabilizethe delivery flow to the pumping system.

a. Prechiller or Chiller-HPLC Pump Assembly

Generally, the prechiller or chiller 200 utilizes dual refrigerationcircuits with tube-in-tube heat exchangers that allow for heat exchangewithout an intervening heat exchange medium. FIG. 3A illustrates a blockdiagram of the two circuit cascade refrigeration heat exchanger presentin the chiller. The two circuits are a low temperature refrigerationcircuit 220 and a high temperature refrigeration circuit 270. In suchsystems, the two circuits are thermally coupled at the condenser 213 ofthe low temperature circuit. In fact, the condenser of the lowtemperature circuit is the evaporator 211 of the high temperaturecircuit. To further simplify the concept, the low temperaturerefrigeration circuit 220 in the chiller is used to super chill the CO₂202 flow to its target temperature and the high temperaturerefrigeration 270 circuit is used to remove the heat from the lowtemperature circuit.

CO₂ flow enters the evaporator of the low temperature circuit at bottlepressure/temperature. Said evaporator is a tube-in-tube heat exchanger210 with an inner tube 214 made of AISI Type 316 stainless steel orsimilar metal suitable for exposure to CO₂. Other materials of use forthe inner tube include without limitation copper, brass, and Type 304stainless steel. Heat is removed from the CO₂ by the flow of cryogenicrefrigerant in the outside tube 212 which is made of copper andsurrounds the inside tube. The heat exchanger is set up as a counterflow heat exchanger for greater efficiency.

The low temperature circuit is used to pull heat from the CO₂ flow tochill it to the required temperature. A cryogenic refrigerant enters thesuction side of the compressor 222 and is discharged at a higherpressure. The compressor may be a 1.4 CC model by Aspen. Work is done bythe compressor to increase the pressure of the cryogenic refrigerant207, which raises its temperature. The cryogenic refrigerant then exitsthe compressor on the discharge (high pressure) side and enters the lowtemperature circuit condenser. The low temperature circuit condenser isthe same unit as the high temperature circuit evaporator. The condenseris a tube in tube heat exchanger. The cryogenic refrigerant flowsthrough the inside tube 264 of the heat exchanger from an inlet 266 toan outlet 268. Heat is removed from the cryogenic refrigerant by aconventional refrigerant 209 flowing in the outside tube 262 whichsurrounds the inside tube. This heat exchanger is arranged as a counterflow heat exchanger for greater efficiency. After having the heatremoved, the cryogenic refrigerant flows through a moisture indicator290′, and then a dryer 229 which has a built in service port. This isthe high pressure side service port. After the dryer, the cryogenicrefrigerant flows through an expansion valve 228. In this case, theexpansion valve is a coiled length of capillary tube. When the cryogenicrefrigerant exits the expansion valve, it is returned back to a lowpressure state, which reduces the temperature before it enters the lowtemperature circuit evaporator 211. The cryogenic refrigerant 207 flowsthrough the outside tube of the evaporator and removes heat from the CO₂202 flowing through the inside tube which it surrounds. Upon exit, thecryogenic refrigerant flows through a moisture indicator 290″ and aservice tee 299″ before returning to the suction side of the lowtemperature circuit compressor 222. This cycle is continuous. The lowtemperature circuit may also comprise an accumulator tank 229 and valve225 for accumulation of cryogenic refrigerant 207.

The high temperature circuit uses a similar flow path with one majordifference. The condenser of the high temperature circuit is a fancooled liquid to air heat exchanger. In the high temperature circuit, aconventional refrigerant 209 enters the suction side of the compressor272 and is discharged at a higher pressure. The change in pressure isaccompanied by a rise in temperature. The refrigerant then flows intothe condenser 274 where forced air is used to remove heat by an airmoving device 276, such as a fan. This heat is transferred to theatmosphere and out of the system. The refrigerant then flows through amoisture indicator and a dryer 294 with built in service port beforegoing through the expansion valve. On exit of the expansion valve 278the refrigerant is returned to a lower pressure and thus lowertemperature. The refrigerant then enters the evaporator 261. Here therefrigerant for the high temperature circuit absorbs heat from the lowtemperature circuit in a tube-in-tube heat exchanger 260. Thetube-in-tube heat exchanger 260 acts as a condenser 263 for thecryogenic refrigerant 207. Upon exit the refrigerant goes through aservice tee and a moisture indicator before returning to the suctionside of the compressor. This cycle is continuous.

To summarize the two circuit cascade system, it is easiest to follow thetransfer of heat into and then out of the system. In the case of thechiller 200, heat is brought into the system by a stream of CO₂. Theremoval of heat from the CO₂ is the ultimate goal of the system. Thisheat is removed by the evaporator of the low temperature circuit. Thelow temperature circuit is then used to transfer heat to the hightemperature circuit. This happens in the high temperature circuitevaporator which is also the low temperature circuit condenser. In thefinal stage of heat transfer, the high temperature circuit transfersheat out of the system and into the atmosphere in the high temperaturecircuit condenser. In short, heat cascades from the CO₂ to the lowtemperature circuit then the high temperature circuit and finally theatmosphere.

FIG. 3B illustrates the connection of the chiller 200 to a traditionalHPLC type SCF Pump 280 with the addition of post-pump heater 284 tobring the fluids up to operational temperatures.

b. Embodiments of Prechiller or Chiller

In varying embodiments, the chromatography system comprises a prechilleror chiller, as described herein, upstream of a pump to cool thesupercritical fluid sufficiently such that it can be pumped through astandard off-the-shelf, commercially available flash chromatography orhigh performance liquid chromatography (HPLC) pump. The prechillerimproves the pumping performance (e.g., the consistency) for asupercritical fluid, e.g., carbon dioxide such that system mass flowrates are proportionate to pump speed.

In varying embodiments, the prechiller cools the liquid phasesupercritical fluid (e.g., CO₂) to a temperature of about −5° C. orless, e.g., −10° C., −15° C., −20° C., −25° C., or less, e.g., but abovethe triple point temperature, e.g., above about −55° C., e.g. to about−40° C., −45° C. or −50° C. Such supercooling or extreme subcoolingreduced in much improved pumping performance. The pumping mass flow ratewas linearly related to speed and repeatability. In varying embodiments,the prechiller subcools the supercritical fluid (e.g., CO₂) from anambient condition of nearly 25° C. to approximately −10° C. or lowertemperatures. In varying embodiments, the system employs a 2-stagerefrigerant on refrigerant chiller system to cool and liquefy the gasphase supercritical fluid. In varying embodiments, the system does notdirectly or separately cool the pump heads.

This minimum of 35° C. temperature reduction resulted in thesupercritical fluid (e.g., liquid phase CO₂) conditions more closelyresembling an incompressible fluid, such as water. A completelyunmodified and standard HPLC pump can then be used to pump thesupercritical fluid (e.g., liquid phase CO₂) under very linearconditions. Such behavior is highly desirable for application includingsupercritical fluid extraction, supercritical fluid solid phaseextraction, supercritical fluid flash chromatography, and supercriticalfluid chromatography.

The supercooling prechiller enables standard and cost effective HPLCpumps to be used in supercritical fluid applications with highlylinearly mass flow rates with the need for either elaborate compensationalgorithms, sensor feedback systems involving compensation via a loss inweight measurement of the supply cylinder, direct compensation via amass flow measure (e.g., coriolis mass meter), or the need for a boosterpump to stabilize the flow.

4. Pressure Equalizing Vessel

a. Introduction

Pressure equalization assemblies and methods of use are provided. Morespecifically, provided is a pressure equalization assembly that enablesthe use of low-medium pressure columns for flash chromatography in ahigher pressure supercritical fluid chromatography application. Thepressure equalization assemblies allow the attachment of commerciallyavailable chromatography columns or cartridges to the cap of the vessel,e.g., via a luer lock fitting, and seals on the other end using anO-ring or gasket that is captured axially by the cap and tapered stem ofsaid column. Sample stream pressure going through the column is balancedby external pressure applied to the same column to maintain a pressuredifferential that is less than the standard operating pressure of thecolumn. In doing so, it ensures that the columns can be used withoutfailure for high pressure supercritical fluid chromatography.

b. Embodiments of the Pressure Equalizing Vessel

In varying embodiments, the supercritical fluid chromatography systemcomprises a pressure equalizing vessel. The pressure equalizing vesselis designed to allow the use of commercially available or off-the-shelflow to medium pressure columns (e.g., in the range of about 14-200 psi)traditionally used in flash chromatography at the higher pressures (inthe range of about 1000 psi to about 10,000 psi, e.g., in the range ofabout 1500-2000 psi) used in Supercritical Flash Chromatography. Thepressure equalizing vessels described herein allow the use of moreeconomical pre-packed disposable columns in Supercritical FlashChromatography, rather than expensive high pressure columns that must bere-packed by the user.

The pressure equalization vessel described herein utilize pressureequalization to allow the low pressure columns to exceed their ratedburst pressures. This is accomplished by pressurizing the outside of thecolumn to a level that ensures that the pressure differential betweenthe flow through the inside of the column and the equalizing pressure onthe outside of the column remains within the rated pressure of thecolumn. For example, if a column is rated at 200 psi normal operatingpressure, and the user desired to run at higher pressure ranges of about1000 psi to about 10,000 psi, e.g., 1500-2000 psi, the system wouldensure that the equalizing pressure is within 200 psi of workingpressure. Testing has proven this to be effective at preventing failureof the columns due to overpressure.

The pressure equalization system allows the attachment of commerciallyavailable or off-the-shelf flash chromatography columns to the cap ofthe vessel via a luer lock fitting, and seals on the other end using anO-ring or gasket that is captured axially by the cap and tapered stem ofsaid column. The pressure equalizing vessel is compatible for use withany commercially available pre-packed flash chromatography cartridge,including without limitation cartridges made by Grace (grace.com),Silicycle (silicycle.com), Biotage (biotage.com), Teledyne-ISCO(isco.com), Buchi (buchi.com), Interchim Inc. (interchiminc.com), andAgilent (agilent.com). The pressure equalizing vessel does not limit thesize of the inner column cartridge that can be used, but is designed toadjust and accommodate to the chromatography cartridge appropriate for adesired separation. In varying embodiments, the inner column can containin the range of from about 4 grams to about 350 grams stationary phasemedia, e.g., 4 grams, 8 grams, 12 grams, 20 grams, 80 grams, 120 gramsor 330 grams stationary phase media. In varying embodiments, the innercolumn comprises a diameter in the range of about 0.5 inches to about3.5 inches and a column length in the range from about 3.5 inches toabout 11 inches. Illustrative diameter and length sizes of the innercolumn include without limitation 0.94 inches diameter.times.3.85 incheslength (4 grams stationary media); 1.3 8 inches diameter.times.4. 60inches length (12 grams stationary media); 1.77 inchesdiameter.times.6.43 inches length (40 grams stationary media); 1.99inches diameter.times.9.50 inches length (80 grams stationary media);2.18 inches diameter.times.10.31 inches length (120 grams stationarymedia); or 3.39 inches diameter.times.10.55 inches length (330 gramsstationary media).

Sample stream pressure going through the column is balanced by externalpressure applied to the same column to maintain a pressure differentialthat is less than the standard operating pressure of the column. Indoing so, it ensures that the columns can be used without failure forhigh pressure supercritical fluid chromatography.

FIG. 8 illustrates an assembly drawing of external views of a productionprototype of the chiller 800 configured within an enclosure 801 having apower connection 803 and having bulkhead connections 805 and 806.

FIG. 9 illustrates an assembly drawing of internal views of a productionprototype of the chiller 900. Fan-cooled heat exchanger 902.Tube-in-tube heat exchanger 905. Sight glass; moisture indicator 912.Sight glass to service Tee Tube 914. Access valve 915. Service Tee tosuction inlet tube 916. Capillary tube winding 921. Union elbow withtube fitting 922. Union coupling with tube fitting 924. Cryo compressorsuction inlet tube 927. Liquid gas outlet tube 928. High capacitycompressor drive 930. Mounting bracket 931. Drive board-compressor 932.1.4 CC compressor 933.

FIG. 10 illustrates an assembly drawing of internal views of aproduction prototype of the chiller 900. Fan mounting bracket 904.Reducing coupling 906. Compressor to condensing tube 907. Solderconnection; copper fitting 908. 90 degree long elbow solder connection909. Condenser to sight port tube 910. Copper tube, 45 degree elbow 913.90 degree long elbow solder connection 917. Dryer, liquid line withservice port 918. Down tube—condenser side 926. Liquid gas inlet tube929. Power supply 934.

FIG. 11 illustrates a cross sectional view of the column system. Itshows the pressure containment vessel 1170, the medium pressure column1190, and the methods for attaching the column to the vessel itself. Thefittings of the pressure equalizing vessel can be readily adjusted toaccommodate the inner column being used, wherein the standard inputfitting accommodates a female luer lock on the inner column and thestandard output fitting accommodates a male slip fitting on the innercolumn. In the embodiment depicted in FIG. 11, a luer lock connectionprovided on the supercritical fluid (e.g., CO₂) plus optional co-solventinlet 1140 of the column seals the outside pressure from the samplestream pressure. The luer lock adapter 1150 is shown as a threadedadapter in this print, but may also be an integral machined part of thevessel cap, or also a welded adapter. On the other end of the column,the outside equalizing pressure, and the sample stream pressure aresealed from each other using an O-ring 1160 or gasket, on the outside ofthe column stem. The cap of the vessel has a shelf to capture saidgasket and the column stem is tapered so that it also helps capture thegasket in position by providing an axial force. This tapered stem andthe luer lock on the opposite end are typical of industry standardlow-medium pressure columns.

FIG. 11 also shows the inlet connection for the sample stream 1140. Thisstream typically is composed of a supercritical fluid (e.g., CO₂),optionally a co-solvent, and the sample to be separated. The fittingshown is a high pressure compression fitting made to seal on the outsidediameter of appropriately sized high pressure tubing. The same type offitting is used for the Sample stream outlet 1110, and the pressureequalizing inlet 1120. The pressure equalizing medium will typically bea supercritical fluid (e.g., CO₂).

FIG. 12 illustrates a cross sectional view of an example supercriticalflash chromatography system 1200 with the pressure equalization systemincorporated. FIG. 12 illustrates the typical input of supercriticalfluid (e.g., CO₂) 1220 and input for co-solvent 1240, and shows how thesystem equalizes pressure in this case. Input flow of supercriticalfluid (e.g., CO₂) is split, one direction serves as the pressureequalizing fluid, and the other direction is used in conjunction withco-solvent to flow with the sample through the column. The systempressure is controlled by a back pressure regulator.

The check valve 1250 after the input tee for supercritical fluid (e.g.,CO₂) ensures that the pressure is typically greater on the outside ofthe low pressure column. This means that if any leaks were to occur, theleaks would occur from outside equalizing fluid, into the column. Thisprotects the valuable samples being separated from being lost.

5. Cyclonic Separator

a. Introduction

In varying embodiments, the supercritical fluid chromatography systemcomprises a cyclonic separator. The cyclonic separator is designed toefficiently and effectively separate sample molecules from a liquidphase or gas phase stream of a supercritical fluid, e.g., CO₂. Theseparator is designed to accept tangential input flow, e.g., via tubecompression fitting, allowing the separator to accept typical industrystandard tubing. Using a tangential inlet, the flow is channeled in acyclonic flow around the separator to separate the molecules from thegaseous flow by centrifugal force. The separator deposits the samplemolecules conveniently into an attached sample collection jar, and canbe completely disassembled for complete cleaning. To ensure anymolecules not successfully separated by the centrifugal forces of thecyclone are not released to atmosphere, a sintered filter of anappropriate size (e.g. having a porosity grade of G-5, or a pore size inthe range of about 1-16 microns) can be pressed into the exit of thecyclone, allowing only the gaseous flow to escape.

b. Embodiments of the Cyclonic Separator

The herein described cyclonic separators are designed to separatemolecules from a gas phase supercritical fluid (e.g., CO₂) flow andcollect the molecules in a sample jar. In varying embodiments,separation procedures are performed at flow rates in the range of about10-300 ml/min, e.g., about 250 ml/min, and at pressures in the range ofabout 1000-10,000 psi, e.g., about 1500-2000 psi or about 1,750 psi. Thecyclonic separators can be used within a pressurized chromatographysystem and in fluid communication with a sample stream using compressionfitting adapters and can be vented to atmosphere directly, or by hookingup a hose to the outlet. All materials of construction are suitable foruse with corrosive solvents.

Other forms of cyclonic separators have been used in the past to attemptto separate a desired sample from CO₂/co-solvent streams in thesupercritical fluid extraction products. These have been much cruder,simpler devices typically consisting of an inlet tube that would bringthe fluid/particle stream into a collection vessel at 90 degrees, theproduct stream would circulate around the interior diameter of thecollection vessel and the particulate products and modifier co-solventswould drop out and settle at the bottom of the collection assembly andthe gaseous SCF CO₂ would vent through and an outlet tube. The problemwith these devices was always the loss of desired product to the fluidgaseous stream on the outlet. This was because none of the devices weredesigned to form a true cyclonic flow, nor were they equipped withproper filtration on the outlets. By contrast, with the presentlydescribed cyclonic separators, a cyclonic flow path is induced in whichthe gas is forced into rotational flow around the exit tube facilitatedby the tangential inlet, and is then forced into a downward spiral intowards the low pressure region by the conical section. The low pressureregion is in the middle of the volume where the exit is located.

FIG. 13 illustrates an overall assembly drawing of the cyclonicseparator and collection assembly 1300. The cyclone body 1301 isconnected to the fluid stream using a National Pipe Thread(NPT)×compression adapter. In this illustrated assembly, the compressionfitting is sized for ⅛″ tube and the NPT fitting is 1/16″. Compressionfittings 1304 may be sized in the range of about 1/16 inches to about ¼inches find use. The cyclone cap threads into the top of the cyclonebody and seals against an O-ring. This ensures that pressure is not lostthrough the threads. The cap 1302 has a sintered filter 1307 pressedinto the exit to ensure that any sample molecules that may not have beenseparated by the vortex flow are captured and not released toatmosphere. Pore sizes of the sintered disc can be sized for particularcompounds. In the illustrated iteration, sintered filter having aporosity grade G-5 is used (1-16 microns pore size). In varyingembodiments, sintered filters with G-0 to G-5 porosity grade find use(G5=pore size in the range of about 1-16 microns; G4=pore size in therange of about 1 0-16; G3=pore size in the range of about 16-40 microns;G2=pore size in the range of about 40-100 microns; G1=100-160 microns;and G0=pore size in the range of about 160-250 microns).

The cyclone body can be configured to be adapted to many standardcollection jars 1305. In the embodiment illustrated in FIG. 13, a 500 mLglass collection jar is used. The cyclone body can have a threadedbottom exit for attachment and sealing to the collection jar. The cap ofthe jar can have a through hole, which allows the cyclone body to besecured to the cap using a nut. This connection can be sealed using anO-ring 1303, as illustrated. Electrical panel nut is 1306 andcompression fitting is 1304.

FIG. 14 illustrates the result of a Computational Fluid Dynamics (CFD)streamline study done to optimize the geometry of the cyclone at 250mL/min @1,750 psi of supercritical CO₂ flow. When the CO₂ flow reachesthe cyclone, it is no longer at such high pressures because the cycloneis open to atmospheric pressure. Because of this, the mass flow rate wascalculated and then used to determine the velocity of the streamentering the cyclone. The shapes utilized have been done so to properlyfunction with the parameters of the particular CFD program. Though theappearance may differ slightly from the assembly in FIG. 13, theinternal geometry of the cyclone body is the same. The stream linespictured illustrate the path and velocity of the fluid flow. The colorsvary from red to blue, with red indicating the highest stream velocity,and blue indicating the lowest stream velocity. Most importantly, FIG.14 illustrates the downward spiral, substantially non-overlapping streamlines typical of an optimized cyclonic separator.

As illustrated in FIG. 14, flow enters the cyclone body tangential tothe inside diameter. The flow then begins to rotate around the exit tubeof the cyclone. The centrifugal forces exerted on the molecules in thestream lines send the molecules outwards to the wall of the cyclonebody, where a boundary layer keeps the streamlines from recollecting themolecules. The molecules are then free to fall to the bottom of thecollection assembly. As the stream lines travel to the bottom of thecyclone body and hit the conical section, the velocity slows and thepressure increases. This forces the streamlines up the exit tube whichis a low pressure escape from the higher pressure conical section.

FIG. 15 illustrates the manufacturing print released to the machine shopfor the current revision of the cyclone body. All dimensions andinformation pertinent to the manufacturing of the part are present. Inthe illustrated embodiment, the threads used to secure the cap to thebody are the 1″-24 Class 2-B threads. Thread size is determined by thebody of the cyclone, wherein the thread size and conformation areselected to withstand pressure and secure the cap. Generally, thethreads are larger than the inside diameter of the cyclone body. In theillustrated embodiment, the body is secured to the collection vesselusing a ⅜″-32 National Extra Fine (NEF) thread. The function of thecyclone is not dependent on these threads, they were selected to aid inmanufacturing and assembly.

FIG. 16 illustrates configurations of the cyclone cap. In theillustrated embodiment, the cyclone cap is secured to the cyclone bodyvia screw threads. The ledge at the top the cap allows for a sinteredfilter to be pressed into the cap.

FIG. 17 illustrates the internal dimensions of the cyclone. Thesedimensions were honed by using CFD visualization of streamlines, asillustrated in FIG. 14. Dimensions important to the functionalityinclude the ratio of the diameter of the outer circumference to theinner diameter of the mid-height of the funnel in the range of about 3to about 4, e.g., about 3.5. In varying embodiments, the funnel has anangle in the range of about 30 degrees to about 60 degrees, e.g., in therange of about 35 degrees to about 55 degrees, e.g., an angle of about30 degrees, 35 degrees, 40 degrees, 45 degrees, 50 degrees, 55 degrees,60 degrees. The illustrated embodiment depicts a 0.875 diameter to 0.250diameter ratio (e.g., a ratio of 3.5) along with a 40-degree funnelangle. A further important dimension includes the dimensions of theprotrusion at the bottom of the cap. Generally, the depth of protrusionat the bottom of the cap extends below the tangential inlet. In varyingembodiments, the depth of protrusion at the bottom of the cap extends,e.g., in the range of about 0.5 inches to about 1 inch, e.g., in therange of about, 0.6 inches to about 0.9 inches, e.g., about 0.50, 0.55,0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 or 1.0 inches. This zonerepresents a part of the internal geometry of the cyclone collectionassembly (see, FIG. 16).

6. Methods of Separating Molecules

Further provided are methods of performing high pressure separationand/or extraction procedures using a flash chromatography system,comprising employing the chiller or prechiller, as described above andherein. The chromatography systems comprising a pressure equalizingvessel, as described herein, are useful for the separation of moleculesthat can be separated using liquid chromatography, e.g., flashchromatography employing commercially available off-the-shelf columncartridges and off-the-shelf HPLC positive displacement pumps.Generally, molecules that can be successfully separated when employing asupercritical fluid solvent have a higher density than the supercriticalsolvent, for example, the molecules may have a higher density thansupercritical, liquid phase and/or gas phase supercritical fluid (e.g.,CO₂). In varying embodiments, the molecules to be separated in thepresently described chromatography systems comprising a cyclonicseparator are small organic compounds, peptides, polypeptides, lipids,carbohydrates, nucleic acids and/or polynucleotides. In varyingembodiments, the molecules to be separated can have a molecular weightin the range of about 40 Daltons (Da or 40 gram/mol) to about 1,000,000Da (g/mol), or more, e.g., in the range of about 100 Da (g/mol) to about10,000 Da (g/mol), e.g., in the range of about 100 Da (g/mol) to about5,000 Da (g/mol).

In varying embodiments, the methods entail inputting a sample to beseparated that is dissolved or suspended in a supercritical fluid (e.g.,CO₂), with or without co-solvent, into the inner column of the pressureequalizing vessel assembly. In varying embodiments, separationprocedures are performed at flow rates in the range of about 10-300ml/min, e.g., about 250 ml/min, and at pressures in the range of about1000-10,000 psi, e.g., about 1500-2000 psi or about 1,750 psi. Theinterspace of the pressure equalizing vessel surrounding the innercolumn is also filled with supercritical fluid at a pressure such thatthe pressure differential between the pressure within the interspace andthe pressure within the inner space of the inside column is less thanthe pressure rating of the inner column (e.g., less than about 14-200psi). Molecules in the sample are separated according to well-knownprinciples of liquid chromatography using commercially available andoff-the-shelf flash chromatography cartridges or columns packed withsolid phase media commonly used in the art.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1: Mass Flow Rates Vs. Temperature Employing the Chiller

FIG. 4 illustrates test data for mass flow rate vs. temperature at 250rpm pump speed. The data is focused around the temperature of −10° C.where pumping consistency increases and mass flow rate and pumpingefficiency also begin to increase with decreasing temperatures. At anaverage temperature of 0.3° C. average mass flow rate was 0.0433 kg/minwith a standard deviation of 0.0024. At an average temperature of −7.95°C., average mass flow rate was 0.0447 kg/min with a standard deviationof 0.0030. At an average temperature of −14.75° C., average mass flowrate was 0.0487 kg/min with a standard deviation of 0.0018. At anaverage temperature of −23.53° C. average mass flow rate was 0.0525kg/min with a standard deviation of 0.0004. At an average temperature of−30.78° C. average mass flow rate was 0.0546 kg/min with a standarddeviation of 0.0005. All tests were performed at a constant RPM of 250,with a target pressure of 2,000 psi and set point flow rate of 13mL/min. The data shows a trend of increasing mass flow rate below −10°C. and decreased variation.

FIG. 5 illustrates test data for mass flow rate vs. temperature at 500rpm pump speed. The data is focused around the temperature of −10° C.,where pumping consistency increases and mass flow rate and pumpingefficiency also begin to increase with decreasing temperatures. At anaverage temperature of 1.3° C., average mass flow rate was 0.0847 kg/minwith a standard deviation of 0.0104. At an average temperature of −9.23°C., average mass flow rate was 0.1067 kg/min with a standard deviationof 0.004 7. At an average temperature of −17.88° C., average mass flowrate was 0.1113 kg/min with a standard deviation of 0.0069. At anaverage temperature of −25.28° C., average mass flow rate was 0.125kg/min with a standard deviation of 0.0005. At an average temperature of−30.37° C. average mass flow rate was 0.132 kg/min with a standarddeviation of 0.0005. All tests were performed at a constant RPM of 500,with a target pressure of 2,000 psi and set point flow rate of 33mL/min. The data shows a trend of increasing mass flow rate below −10°C. and decreased variation.

FIG. 6 illustrates test data for mass flow rate vs. temperature at 750rpm pump speed. The data is focused around the temperature of −10° C.where pumping consistency increases and mass flow rate and pumpingefficiency also begin to increase with decreasing temperatures. At anaverage temperature of −0.13° C., average mass flow rate was 0.158kg/min with a standard deviation of 0.003. At an average temperature of−8° C., average mass flow rate was 0.173 kg/min with a standarddeviation of 0.0047. At an average temperature of −16.2° C. average massflow rate was 0.181 kg/min with a standard deviation of 0.001. At anaverage temperature of −23.8 T C, average mass flow rate was 0.192kg/min with a standard deviation of 0.001. At an average temperature of−31.65° C., average mass flow rate was 0.201 kg/min with a standarddeviation of 0.0004. All tests were performed at a constant RPM of 750,with a target pressure of 2,000 psi and set point flow rate of 70mL/min. The data shows a trend of increasing mass flow rate below −10°C. and decreased variation.

Example 2: Separation of Aceptophenone and Methyl Paraben

0.1 grams of Aceptophenone and 0.1 grams of Methyl Paraben weredissolved in 2 mls of Ethyl Acetate. This sample was injected into thesample loop of the SCF CO₂ Flash Chromatography unit with a flow rate of50 mls/minute of SCF CO₂ and 10 mls/min of Ethyl Acetate at 1750 psi(120 Bar) and 50° C. These materials were separated through the 40 gramsilica cartridge column and collected in cyclonic separators with a99%+efficiency.

The SCF CO₂ Flash unit, for the purposes of the present and followingexamples, was operated at 50 mls/minute SCF CO₂ and 10 mls/minute up to1 7.5 mls/minute of modifier co-solvent in an isocratic or gradient modeat 1750 psi (120 bar) and 50° C. The SCF CO₂ Flash Chromatography unitis capable of operation up to 2500 psi (175 bar) with a SCF CO₂ flowrate of 250 mls/minute and co-solvent modifier flow rate of up to 100mls/minute with a maximum operational temperature of 100°. TheUltra-Chiller cools the CO₂ liquid coming from the supply tank fromambient temperature down to −25° to −30° which allows for efficient andaccurate pumping of the SCF CO₂. Once the SCO₂ liquid has been pumped,it flows through a pre-heater that brings the fluid from the −25° to−30° pump exit temperature up to operation temperatures of up to 100° C.The fluid streams (a supercritical fluid, e.g., supercritical CO₂, andCo-Solvent modifier) flow through a static mixer that ensures thehomogeneous mixing of the fluids for delivery to the column assembly.Sample introduction into the unit occurs in two modes: samples dissolvedin solvent up to 5 mls in size are introduced through a sample injectionloop, larger samples can be introduced through a column injectionmanifold (reaction mixture is evaporated onto a course silica gel thatis placed in the column assembly for injection).

For the purposes of this work a 40 gram W. R. Grace traditional flashcartridge was used (Grace Reveleris Silica 40 micron, 40 gram, Lot#09071032, PIN 5146132, Pressure Rating 20 Opsi). However, the pressureequalizing vessel or Column Cartridge Containment Assembly canaccommodate traditional flash cartridges from Grace (4 grams up to 330grams in size) and flash cartridges from other flash chromatographyvendors (Silicycle (silicycle.com), Biotage (biotage.com), Teledyne-ISCO(isco.com), Buchi (buchi.com), etc.). The UV-Vis detector was set to 254nm to detect the fractions coming from the separation column to then becollected in the Cyclonic Separator Assemblies. Each individual peak canbe collected as a pure fraction. The results are shown in FIG. 18.

Example 3: Separation of Aceptophenone and Methyl Paraben

0.1 grams of Aceptophenone and 0.1 grams of Methyl Paraben weredissolved in 2 mls of Ethyl Acetate. This sample was injected into thesample loop of the SCF CO₂ Flash Chromatography unit with a flow rate of50 mls/minute of SCF CO₂ and gradient of 10 mls/min to 17.5 mls/min ofEthyl Acetate at 1750 psi (120 Bar) and 50° C. These materials wereseparated through the 40 gram silica cartridge column and collected incyclonic separators with a 99%+efficiency. The results are shown in FIG.19.

Example 4: Separation of Benzoic Acid and 4-Acetamidophenol

0.1 grams of benzoic acid and 0.1 grams of 4-acetamidophenol weredissolved in 2 mls of Methanol. This sample was injected into the sampleloop of the SCF CO₂ Flash Chromatography unit with a flow rate of 50mls/minute of SCF CO₂ and gradient of 10 mls/min to 17.5 mls/min ofMethanol at 1750 psi (120 Bar) and 50°. These materials were separatedthrough the 40 gram silica cartridge column and collected in cyclonicseparators with a 99%+efficiency. The results are shown in FIG. 20.

Example 5: Separation of Ketoprofen and 4-Acetamidophenol

0.1 grams of ketoprofen and 0.1 grams of 4-acetamidophenol weredissolved in 2 mls of Methanol. This sample was injected into the sampleloop of the SCF CO₂ Flash Chromatography unit with a flow rate of 50mls/minute of SCF CO₂ and gradient of 10 mls/min to 17.5 mls/min ofMethanol at 1750 psi (120 Bar) and 50° C. These materials were separatedthrough the 40 gram silica cartridge column and collected in cyclonicseparators with a 99%+efficiency. The results are shown in FIG. 21.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

7. Alternative Pressurization Methods

Alternative methods (to chilling or pre-chilling) exist to allow anon-polar solvent (e.g., CO₂) to perform high pressure chromatographicseparation and/or extraction at or near supercritical fluid state, as ameans to reduce or eliminate the need for polar (e.g., organic)solvents. One such pressurization method is disclosed in U.S. Pat. No.8,215,922 to Berger et. al., incorporated herein by reference, wherein aset of pumps is described for pressurizing “compressible fluids” (e.g.,CO₂) to enable flow for an ultrahigh performance chromatographic systemor supercritical fluid chromatography system. The '922 Berger patentdoes not, however, disclose a pressure equalization means to allow forthe use of this pressurized supercritical fluid technique withtraditional disposable cartridges as the separation column, which is notdesigned to withstand high pressure. Utilizing the pressure equalizationtechniques described herein, one may utilize the set of pumps disclosedin the Berger '922 patent to pressurize and meter flow of a non-polarsolvent at or near supercritical phase and employ this technique withtraditional disposable cartridge systems.

8. Alternative Separation Methods

Alternative methods (to cyclonic separation) exist to separate andcollect sample molecules from a liquid phase or gas phase stream ofpressurize non-polar solvent (e.g., CO₂) utilizing chromatographicseparation and/or extraction techniques at or near supercritical fluidstate, as a means to reduce or eliminate the need for polar (e.g.,organic) solvents. One such separation method is disclosed in U.S. Pat.No. 6,413,428 to Berger et. al., incorporated herein by reference,wherein a controlled decompression system is disclosed to allow forphased (step-down) pressure reduction or decompression to efficientlycontrol separation and collection of desired sample molecules. The '428Berger patent does not, however, disclose a chilling or pre-chillingtechnique to allow for pressurization of non-polar solvent at or nearsupercritical fluid state and subsequent flow through the system with asingle non-polar solvent pump (unlike the discloser in the Berger '922patent). Utilizing the chilling or pre-chilling techniques describedherein, one may utilize the separation and collection by non-polarsolvent decompression disclosed in the Berger '428 patent tofractionally separate and collect sample molecules from the pressurizedsystem, thereby reducing or elimination the need for polar solvents.

9. Alternative Separation Methods

A Pressure Containment Assembly (PCA) can be employed in the MediumPressure Liquid (Flash) Chromatography. Typical operation pressures ofthese units is 5-50 Bars (75-725 psi). Medium Pressure LiquidChromatography (MPLC) is one of the various preparative columnchromatography techniques for separation of materials. Separation underelevated pressures renders the use of smaller particle size columnpacking supports possible (typical stationary phase particles in thecolumns of size 15-40 μm particle size are used) and increases thediversity of usable stationary phases for the column cartridges. Thisseparation method is now routinely used beside or in combination withthe other common preparative tools (e.g. ISCO, Biotage, and Grace).Using PCA affords significant cost advantage by allowing use of lessexpensive plastic column cartridges.

It is herein disclosed and taught how PCA “pressure balancing” may beused to counter the pressure applied to the solvent flow stream that isthus exerted on the interior of the plastic column cartridge. Using aninert gas (e.g., Nitrogen or Air) one may employ PCA in a traditionalall solvent medium pressure flash chromatography unit.

The control logic for the PCA includes a pressure transducer, locatedupstream of the PCA, reading pressure from the solvent flow stream andcommunicating these pressure readings to a control value capable ofapplying counter-balancing pressure from an inert gaseous source(N2/Air) delivered to the PCA (5-50 bars, 75-725 psi). The pressure ofthe inert gas introduced into the PCA is at or near the same pressureand rate of pressure increase as the solvent flow stream being deliveredto the interior of the column. The PCA may also be decompressed at thesame pressure and rate of pressure decrease as the solvent flow streamexiting the interior of the column at the end of a process run. Thepressure balancing provided through the PCA ensures that the disposableplastic cartridge/separation column will neither burst nor be crushed bya pressure differential exceeding its design limits.

The schematic layout of a medium pressure flash chromatography system2200 according to a preferred embodiment of the invention is disclosedin FIG. 22. The system includes two sources of traditional solvent,solvent source 132 and co-solvent source 122. Each solvent is fed intothe system through a solvent pump, solvent pump 136 and co-solvent pump126. The pumped/pressurized solvents mix within the system at staticmixer 140. Valve 116 determines whether the pressurized solvent mixtureflows to a sample injection vessel 121 (for larger samples) or sampleinjection valve 123. Valve 2222 is position to receive and direct flowstreams exiting sample injection vessel 121 and valve 116. From valve2222, the flow stream is directed to sample injection valve 121. Thus,the example sample injection manifold illustrated in FIG. 22 allows forsample injection into the loop through sample injection valve 123 orthrough a secondary injection column (sample injection vessel 121),intended for larger sample injection. Continuing with the schematiclayout of FIG. 22, once the flow stream exits sample injection valve123, the flow stream mixture now includes the solve, co-solvent andsample. At this point, a pressure reading from a pressure transducerwill dictate the amount of pressurized gas to be released from gaspressure source 2232 (e.g., a pressurized air or N2 gas tank/canister)through control valve 133 to provide a pressure balance into pressurecontainment assembly 2240. The pressure reading may be obtained from atransducer that is part of one of the solvent pump assemblies, such asthe reading from co-solvent pump 114 as illustrated in FIG. 22.Alternatively or additional, the pressure reading may be obtained for aseparate pressure transducer such as pressure transducer 131, again, asillustrated in FIG. 22. Valve 133 may further be capable of obtainingpressure readings from multiple transducer sources. The type of gas tobe used at gas pressure source 132 should be an inert gas, such as airor nitrogen (N2). Further describing the schematic layout of FIG. 22,pressure containment assembly 2240 is configured to hold plasticcartridge/column 141, which may be an industry standard disposablecartridge containing silica gel for chromatographic separation. Thesestandard plastic cartridges are not designed to withstand elevatedpressures, thus pressure containment assembly 2240 allows for balancing,to an acceptable differential, the internal and external pressuresapplied to the cartridge walls during system operation. Concluding thedescription of FIG. 22, solenoid valve vent 134 is utilized to regulatethe depressurization of pressure containment assembly 140. Venting ofthe pressurized gas at solenoid valve vent 134 is dictated by a pressurereading from a pressure transducer. The pressure reading may be obtaineda pressure transducer that is part of one of the solvent pumps or,alternatively, from a separate pressure transducer such as pressuretransducer 131 or pressure transducer 151, as illustrated in FIG. 22.The valves may further be capable of obtaining pressure readings frommultiple transducer sources. Without regulation of the gas pressure bycontrolled venting at solenoid valve vent 134, plastic cartridge/column141 may be crushed (implode) due to exceedingly high pressure outsidethe cartridge relative to the sample stream pressure inside thecartridge. Similarly, without regulation of the gas pressure controlledby valve 133 receiving pressure reading input from transducer 131, thecartridge may explode due to exceedingly high sample stream pressureinside the cartridge relative to the pressure outside the cartridge. Thesample collection stream outlet 153 is shown

Turning to FIG. 23, this figure illustrates a cross sectional view ofthe pressure equalization system 2300, i.e., pressure containmentassembly 2340, of an exemplary medium pressure flash chromatographysystem, the assembly incorporating plastic cartridge/column 1190. FIG.23 illustrates the location of the input, inlet 1140, of a combinedsolvent and sample flow stream, and shows how the system equalizes(i.e., counterbalances) the pressure of this flow stream with a pressureequalizing gas input from inlet 1120. The fitting shown in FIG. 23 is ahigh pressure compression fitting made to seal on the outside diameterof appropriately sized high pressure tubing. The same type of fitting isused for sample stream outlet 1110, and pressure equalizing inlet 1120.The pressure equalizing medium will typically be an inert gas (e.g., airor N2).

A combined solvent and sample flow stream is introduced at inlet 1140.Input of the combined solvent and sample flows through the contents ofcartridge 1190. The internally exerted pressure of the flow streamwithin cartridge 1190 is balanced by the externally exerted pressure ofthe pressure equalizing gas 1121 on the exterior 1191 of cartridge 1190.System pressure is controlled by pressure transducer readings.Transducers may be located upstream of inlet 1140 or downstream ofoutlet 1110, or both.

The pressure of the equalizing gas input at inlet 1120 may be moderatelyhigher on the outside of cartridge 1190, but still within pressuretolerance rating of cartridge 241, as compared to the pressure from theflow stream on the inside 1193. By ensuring that any moderatedifferential favors higher external pressure, any leaks to cartridge1190 would result in inert gas movement into cartridge, rather than aloss of the combined solvent and sample flow stream out of cartridge.This protects valuable sample material from being lost.

10. Combination of Various Methods

Certain components and elements of the systems and methods disclosedherein may be combined in various innovative ways.

In one such example, the system may be a chromatography system withreduced solvent volumes using multiple pumps as pressure sources on flowstreams containing highly pressurized gas, compressible liquid, orsupercritical fluid, comprising: a primary pump for pumping a relativelycompressible solvent fluid flow stream; a secondary pump for pumping afirst relatively incompressible fluid flow stream; a mixing device thatcombines said first and second flow streams into a combined flow stream;an injection device that can introduce samples or solutions into any ofsaid flow streams; and a pressure equalizing vessel downstream of saidinjection device, wherein the pressure equalizing vessel comprises aninner chromatography column comprising stationary phase media and anouter column that cylindrically surrounds the length of the innercolumn, wherein no part of the inner column is exposed to full internalpressure without balancing external equalizing pressure. Thischromatography system may further include a pressure equalizing vesselthat has interspace of a width of at least 1 mm between the innerdiameter of the outer column and the outer diameter of the innerchromatography column, and/or an outer column that withstands pressuresof at least about 500 psi and even up to 10,000 psi.

In another such example, the system may be a chromatography system withreduced solvent volumes using multiple pumps as pressure sources on flowstreams containing highly pressurized gas, compressible liquid, orsupercritical fluid, comprising: a primary pump for pumping a relativelycompressible solvent fluid flow stream; a secondary pump for pumping afirst relatively incompressible fluid flow stream; a mixing device thatcombines said first and second flow streams into a combined flow stream;an injection device that can introduce samples or solutions into any ofsaid flow streams; a chromatography separation column, downstream ofsaid mixing device and that can receive said combined flow stream; acyclonic separator downstream of said injection device, to allow forsamples to be collected and recovered, wherein the cyclonic separatorcomprises a cyclone body comprising an inner surface, an outercircumference, a top outlet, a tangential inlet and a bottom outlet.This chromatography system may further include a cyclonic separatorwherein (i) the top portion of the inner surface of the cyclone bodycomprises screw threads, (ii) the middle portion of the inner surface ofthe cyclone body is cylindrical, and (iii) the bottom portion of theinner surface of the cyclone body comprises a funnel. Thischromatography system may further include a cyclonic separator whereinthe funnel has an angle in the range of about 30 degrees to about 60degrees and wherein the ratio of the diameter of the outer circumferenceto the inner diameter of the mid-height of the funnel is in the range ofabout 3 to about 4. This chromatography system may further include acyclonic separator wherein the cyclone body comprises a cap comprising asintered filter and screw threads, and wherein the screw threads of thecap interlock with the screw threads on the inner surface of the topportion of the cyclone body. This chromatography system may furtherinclude a cyclonic separator wherein the cyclonic separator canwithstand pressures of at least about 1000 psi, and wherein the body isin fluid communication with the cap.

In another such example, the method may be a method of performingchromatography using a disposable cartridge, the method comprising thesteps of: i) providing a pressurized vessel and a disposable plasticflash chromatography cartridge removably attached by a fitting to thevessel, wherein the cartridge is loaded with silica or modified silicagel; ii) transporting a flow stream of gas, liquid, or supercriticalfluid through a first refrigerant circuit wherein the first refrigerantcircuit comprises (a) a first compressor that pumps refrigerant, and (b)a first tube-in-tube heat exchanger in fluid communication with thefirst compressor, wherein the first tube-in-tube heat exchangercomprises an inner lumen and an outer lumen that surrounds the innerlumen, and wherein the refrigerant flows through the outer lumen; iii)further chilling the flow stream by transporting the flow stream througha cryogenic refrigerant circuit in thermodynamic communication with thefirst refrigerant circuit where the cryogenic refrigerant circuitcomprises a second compressor that (A) pumps cryogenic refrigerantthrough the cryogenic refrigerant circuit, and (B) is in fluidcommunication with the first tube-in-tube heat exchanger; wherein thecryogenic refrigerant flows through the inner lumen, and whereincryogenic refrigerant circuit further comprises a second tube-in-tubeheat exchanger in fluid communication with the first tube-in-tube heatexchanger, and wherein the second tube-in-tube heat exchanger comprisesan inner lumen and an outer lumen that surrounds the inner lumen,wherein the cryogenic refrigerant flows through the outer lumen andwherein the flow stream becomes further chilled while flowing throughthe inner lumen; iv) transporting the further chilled flow stream out ofthe inner lumen of the second tube-in-tube heat exchanger; v) injectinga sample into the flow stream; vi) receiving the flow stream and samplein an inlet of the cartridge; and vii) collecting fractions of the flowstream exiting an outlet of the cartridge in a collection tray. Thischromatography method may further include a method wherein the fittingon the disposable plastic flash chromatography cartridge is a luer lockfitting, and/or the disposable plastic flash chromatography cartridgecomprises two ends, a fitting end and a seal end, and/or the seal end issealed to the column using an O-ring; and/or the seal end is sealed tothe column using a gasket, and/or the disposable plastic flashchromatography cartridge has a stationary phase media capacity ofbetween 4 grams and 350 grams. This chromatography method may alsoinclude a method wherein the pressurized vessel is equipped to fit witha range of differently sized disposable plastic flash chromatographycartridges, and/or the cartridge has a diameter ranging in size from 0.5inches to 3.5 inches and a length ranging in size from 3.5 inches to 11inches.

In another such example, the method may be a method of performingchromatography with reduced solvent consumption in adisposable-cartridge chromatographic system, the method comprising thesteps of: i) pumping carbon dioxide through a primary pump to allow thecarbon dioxide gas to reach a supercritical fluid phase and function asa non-polar solvent in a chromatographic system; ii) pumping a polarco-solvent through a secondary pump, wherein the polar co-solventcomprises less than or equal to 20 percent of the total volumeconcentration of the total solvents used in the chromatographic system;iii) mixing the polar solvent and the non-polar solvent into a combinedflow stream; iv) injecting a sample into the combined flow stream,wherein the sample is a material to be chromatographically separated; v)receiving the sample and combined flow stream into a disposable plasticcartridge attached to a pressurized vessel, wherein the cartridge is achromatographic column loaded with silica or modified silica gel; andvi) collecting fractions of the separated sample from the flow streamexiting an outlet of the cartridge in a collection tray. Thischromatography method may further include a method wherein the carbondioxide is pre-chilled and passed through a single primary pump beforethe mixing step, or, wherein the carbon dioxide is not pre-chilled andis passed through a series of primary pumps before the mixing step. Thechromatography method may alternatively include a method using chilledcarbon dioxide in a disposable-cartridge chromatographic system, themethod comprising the steps of: chilling a stream of carbon dioxide gasby transmitting the carbon dioxide through a chiller wherein the carbondioxide output from the chiller is at least about 35° C. lower than thecarbon dioxide input into the chiller; ii) pumping the chilled carbondioxide stream through a single, primary, piston-style positivedisplacement pump; iii) without pumping the chilled carbon dioxidestream through an additional pump, injecting a sample into the carbondioxide stream, wherein the sample is a material to bechromatographically separated; iv) receiving the sample and carbondioxide stream into a disposable plastic cartridge attached to apressurized vessel, wherein the cartridge is a chromatographic columnloaded with silica or modified silica gel; and v) collecting fractionsof the separated sample from the flow stream exiting an outlet of thecartridge in a collection tray. This chromatography method may furtherinclude a carbon dioxide stream that is combined with a separate flowstream of polar solvent constituting 20% or less of the total volumeconcentration of the combined polar solvent and carbon dioxide stream,and/or a carbon dioxide stream that is chilled to a temperature in therange of about −10° C. to about 40°, and/or a carbon dioxide streampumping through a single piston-style positive displacement pump with amass flow rate that is repeatable and proportionate to the pump speed.

In another such example, the method may be a method of separating asample into constituent parts using a high pressure liquidchromatography device with a disposable plastic column cartridge,comprising: inserting the disposable plastic column cartridge into apressure containment area of the chromatography device, wherein thecartridge has inlet and outlet sections and is preloaded with silicagel; pressurizing a flow stream of solvent; introducing a sample intothe pressurized solvent flow stream; maintaining, within a pressurecontainment assembly, a pressure balance between external pressure onthe cartridge applied within the pressure containment area and internalpressure on the cartridge applied through the introduction ofpressurized solvent flow stream and sample mixture; wherein the externalpressure is applied through an inert gas source that is controlled by apressure control value influenced by a pressure transducer reading thepressure of the combined sample and solvent flow stream entering thecartridge, and; wherein the pressure balance in maintained in such a wayso that the difference in pressure between the exterior and interior ofthe cartridge is at all times less than the difference in pressure thatwould cause mechanical warping that would damage a wall of thecartridge.

In another such example, there may be a cooling method comprised of theexpansion of a fluid with a positive Joule-Thomson coefficient from anarea of high pressure to one of lower pressure without capturing orrecompressing the fluid, such as shown in FIG. 24, where expansion isaccomplished by passing the fluid through an expansion device 2408 withone or more orifices or capillaries and the expansion device is placedin close proximity to where the cooling effect is needed (e.g., object2409); the expanded fluid may be passed through larger diameter channelsto increase the residence time and surface area and allow the cold lowpressure fluid to absorb energy from the object being cooled; the largerdiameter channels can be machined directly into the object being cooledfor superior heat transfer. Further efficiencies can be obtained byusing the low pressure fluid as it is exhausted and has approachedtemperature equilibrium with the object being cooled to cool theincoming high pressure fluid before expansion (e.g., optional heatexchanger 2404) to reduce its enthalpy. This can be accomplished with acounter flow or other heat exchanger. The amount of cooling and/ortemperature of the device being cooled can be controlled using feedbackfrom a temperature sensor 2407 placed on the device being cooled 2409. Avalve 2405 from this sensor is used to control (e.g., via temp.controller 2406) the flow into the pressure reduction device 2408. Thiscooling method and systems 2403 utilizing this method will have numerousadvantages such as very compact, low noise, low equipment cost, lowoperating cost, and the ability to reach lower temperatures that manyconventional refrigeration systems. The Joule-Thomson fluid may beexhausted from the system through exhaust 2410. A valve 2402 may controlthe flow of pressurized fluid 2401 into the system

As shown in FIG. 25, an exemplary open loop cooling circuit 300comprises a refrigerant inlet heat exchanger 360 for pre-cooling thecooling fluid 350, such as a refrigerant, prior to expansion and flowinto the primary heat exchanger 310, which may be a tube-in-tube heatexchanger 312. A cooling fluid, such as a fluid having a positiveJoule-Thomson coefficient, may be provided from a compressor 351, suchas a compressed gas cylinder, and flow as source cooling fluid 320 tothe inlet heat exchanger 360. The cooling fluid then flows from theinlet heat exchanger as pre-cooled cooling fluid 354 to the primary heatexchanger 310 to cool an object 390, wherein the cooling fluid expandsthrough an expansion device 326 to reduce the temperature of the coolingfluid. In some cases, the cooling fluid reaches cryogenic temperaturesand is a cryogenic refrigerant in the primary heat exchanger. Thecooling fluid may then exit the primary heat exchanger and return to theinlet heat exchanger as return cooling fluid 327. The object 390 may bea solid or may be in thermal communication with a fluid, such as asupercritical fluid 380 that flows through a conduit 382. Thesupercritical fluid conduit may be the object that is cooled by the openloop cooling system and the supercritical fluid may be part of a flashchromatography system as described herein. The primary heat exchanger310 may comprise one of more channels or conduits for the flow of thecooling fluid 350 therethrough, wherein the cooling fluid adsorbs heatfrom the object to be cooled. The channels or conduits may be machinedin and along a conduit for a fluid, such as the supercritical fluid, toflow therethrough. In addition, the supercritical fluid may flow throughthe inner conduit of the tube-in-tube heat exchanger. The heat exchangermay be configured in a pump or portion of a pump. The cooling fluidpasses through the inlet heat exchanger 360, wherein the inlet coolingfluid is cooled by return cooling fluid flowing back from the primaryheat exchanger 310. The cooling fluid may be at a lower temperatureafter exiting the primary heat exchanger than the source cooling fluid320 or pre-cooled cooling fluid 354 and therefore may be used to reducethe temperature of the source cooling fluid 320 in the inlet heatexchanger to produce the pre-cooled cooling fluid 354. The pre-heatexchanger may comprise a tube-in-tube heat exchanger, wherein the inletconduit 322 or inner conduit, of the source cooling fluid 320 is withinan outer tube or conduit of the return conduit 362 for the returncooling fluid 327. The cooling fluid may flow through the return conduitand then out an outlet 328 or vent. In this open loop cooling circuit360, the cooling fluid is exhausted from the system. The pre-cooledcooling fluid 354 may then flow through an expansion device 326, such asan expansion valve wherein the temperature of the cooling fluid isreduced prior to flowing into the primary heat exchanger 310. In theprimary heat exchanger, the cooling fluid absorbs heat from an object tobe cooled 390, such as a fluid flowing through a conduit. In anexemplary embodiment, this fluid is a supercritical fluid that is usedin a chromatography application. The primary heat exchanger may be atube-in-tube heat exchanger, a plate and frame heat exchanger and thelike. A controller 370 may incorporate a temperature sensor 372 formonitoring the temperature of the object to be cooled and a temperaturecontroller 374 that regulates a valve 324 for the flow of cooling fluidthrough the open loop cooling system 300. The rate of flow of thecooling fluid through the expansion device 326 and into the primary heatexchanger 310 may be controlled by the control system 370. The fluid 380flowing through the primary heat exchanger 310 may be chilled or reducedin temperature from the inlet 384 to the outlet 386 of the primary heatexchanger.

FIG. 26 shows a graph of Joule-Thomson coefficients as a function oftemperature for a number of fluids. As shown helium, hydrogen, nitrogenargon and carbon dioxide all have a positive Joule-Thomson coefficientover a wide range of temperatures including in the cryogenic temperaturerange.

As shown in FIG. 27, a primary heat exchanger 2710 is configured in apump housing 2720. There are a plurality of inlets for the cooling fluidor refrigerant to cool the housing and a fluid pumped thereby. Therefrigerant, such as a fluid having a positive Joule-Thomson coefficientmay flow through an orifice 2750 and then expand into channels 2755 thatextend along the pump housing, such as along the length of the pump head2730. The orifice may be smaller than the channels. An exemplary orificemay have an opening that is about 0.10 mm (0.004 inches), and thechannels may have a cross-length dimension or diameter of about 0.25 mm(0.010 inches). The refrigerant or cooling fluid may then exit the heatexchanger from a cooling fluid outlet 2760 as return cooling fluid thatis used to cool source refrigerant or cooling fluid. Preferably, thecooling circuit may be configured with an orifice opening of 0.10 mm(0.004 inches), followed by a 0.010 inch orifice feeding into thechannels in the pump head of the circuit, where the channels in the pumphead are approximately 6 mm (0.25 inches) in diameter. Larger channelsare also preferable as they have more area available for heat transfer.Alternatively, a capillary coil could be utilized for expansion, withthe coil wrapped around a pump (or other device) needing cooling withoutmodifying the device to have internal channels. The expansion ratio froman orifice opening to an orifice or channel for the Joule-Thomson fluidto expand is at least 2 to 1 and more preferably 5 to 1 to 10 to 1 ormore.

What is claimed is:
 1. A circulator system utilizing the expansion of afirst stream of a refrigerant to cool a second stream of therefrigerant, the system comprising: (i) a pressurized, open-loop circuitwith an inlet portion, a pressurized portion and an expansion portion;(a) the inlet portion comprising a source container of the refrigerant,held at ambient temperature, configured to supply the system with therefrigerant to flow through the system; (b) the pressurized portioncomprising a chiller pump an operational speed within the circuit, thepressurized portion configured to maintain the second stream of therefrigerant in continuous circulation at: (1) a mass flow rate that isrepeatable and proportionate to the operational speed of the chillerpump, (2) a temperature between −5° C. and −30° C., and (3) a continuouspressure of between 500 psi and 10,000 psi; wherein said chiller pumphas a pump head; (c) a heat exchanger comprising: a pump housing; atleast a portion of the pump head confirmed in the pump housing; theexpansion portion comprising an expansion device, comprising: an inletorifice into the pump housing; an outlet orifice from the pump housing;and a channel configured in the pump housing between and connecting theinlet orifice and outlet orifice, through which the first stream of therefrigerant is expanded and cooled, by virtue of the Joule-Thompsoneffect to cool the chiller pump, and wherein the second stream of therefrigerant flowing from the chiller pump is between −5° C. and −30° C.2. The circulator system of claim 1, wherein the refrigerant is selectedfrom the group consisting of hydrogen, nitrogen, argon, carbon dioxide.3. The circulator system of claim 1, wherein the refrigerant is carbondioxide.
 4. The circulator system of claim 1, wherein the refrigerantcirculating within the circuit as a liquid is maintained at atemperature that is warmer than the triple point temperature for theliquid.
 5. The circulator system of claim 1, wherein pressurized portionis configured to maintain a mass flow rate of between 10 milliliters perminute and 300 milliliters per minute of the refrigerant within thepressurized portion.
 6. The circulator system of claim 1, whereinpressurized portion is configured to maintain a mass flow rate of atleast 50 milliliters per minute of the refrigerant within thepressurized portion.
 7. The circulator system of claim 1, wherein thecircuit includes no more than one single pump.
 8. The circulator systemof claim 1, wherein the circuit is configured to prevent the refrigerantfrom evaporating within the pressurized portion.
 9. The circulatorsystem of claim 1, wherein the circuit is configured to prevent therefrigerant from forming condensate within the pressurized portion. 10.The circulator system of claim 1, wherein the chiller pump is apiston-style positive displacement pump.
 11. The circulator system ofclaim 1, wherein the chiller pump is an HPLC—(High Pressure LiquidChromatographytype) pump.
 12. The circulator system of claim 1, whereinthe chiller pump is configured to pressurize the refrigerant within thepressurized portion to between 1,700 psi and 1,800 psi.
 13. Thecirculator system of claim 1, wherein the chiller pump is configured topressurize the refrigerant within the pressurized portion to at least10,000 psi.
 14. The circulator system of claim 1, wherein therefrigerant within the pressurized portion is chilled at least 35° C.lower than the refrigerant in the source container.
 15. The circulatorsystem of claim 1, wherein the expansion device contains at least oneinlet orifice for refrigerant flow and at least one outlet orifice forrefrigerant flow, and the expansion ratio between the at least one inletorifice and the at least one outlet orifice is equal to or greater than5 to
 1. 16. The circulator system of claim 15, wherein the expansionratio is equal to or greater than 10 to
 1. 17. The circulator system ofclaim 1, wherein the pump-pressurized portion of the circuit comprises achromatographic column configured to allow the refrigerant to passthrough a layer of stationary phase media to separate chemicals.
 18. Thecirculator system of claim 17, wherein internal and external pressure onthe chromatographic column is balanced such that pressure differentialon any wall separating the interior of the column from the exterior ofthe column is no greater than 200 psi.
 19. The chiller of claim 18,wherein the first refrigerant stream is an open loop cooling circuit,wherein the first refrigerant is expelled from the circuit after passingthrough an inlet heat exchanger.